Biocompatible polymers, process for their preparation and compositions containing them

ABSTRACT

A process for treating fibroses including administering a therapeutically effective amount of a pharmaceutical composition which includes at least one biocompatible polymer of the following general formula (I): A a X x Y y  wherein: A represents a monomer selected from the group consisting of a sugar or —(O—CH 2 —CH 2 —CO)—, X represents a carboxyl group bonded to monomer A and is contained within a group according to the following formula: —R—COO—R′, in which R is a bond or an aliphatic hydrocarbon chain, optionally branched and/or unsaturated, and which can contain one or more aromatic rings except for benzylamine and benzylamine sulfonate, and R′ represents a hydrogen atom or a cation, Y represents a sulfate or sulfonate group bonded to monomer A and is contained within a group according to one of the following formulas: —R—O—SO 3 —R′, —R—N—SO 3 —R′, —R—SO 3 —R′, in which R is a bond or an aliphatic hydrocarbon chain, optionally branched and/or unsaturated, and which can contain one or more aromatic rings except for benzylamine and benzylamine sulfonate, and R′ represents a hydrogen atom or a cation, a represents the number of monomers A such that the mass of the polymers of formula (I) is greater than approximately 5,000 da, x represents a substitution rate of the monomers A by the groups X, which is between approximately 20 and 150%, and y represents a substitution rate of the monomers A by the groups Y, which is between approximately 30 and 150%.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.12/212,093, filed Sep. 17, 2008, which is a Continuation of applicationSer. No. 10/695,574, filed Oct. 28, 2003, which is a Divisional ofapplication Ser. No. 09/765,788, filed Jan. 19, 2001 now U.S. Pat. No.6,689,741, issued Feb. 10, 2004, which is a continuation ofInternational Application No. PCT/FR99/01774, with an internationalfiling date of Jul. 20, 1999, which claims priority to French PatentApplication No. 98/09309, filed Jul. 21, 1998, the disclosure of theprior applications are incorporated in its entirety by reference.

RELATED APPLICATION

This application is a continuation of application Ser. No. 10/695,574,filed Oct. 28, 2003, which is a divisional of application Ser. No.09/765,788, filed Jan. 19, 2001 (now U.S. Pat. No. 6,689,741, issuedFeb. 10, 2004), which is a continuation of International Application No.PCT/FR99/01774, with an international filing date of Jul. 20, 1999,which is based on French Patent Application No. 98/09309, filed Jul. 21,1998.

TECHNICAL FIELD

This invention pertains to new biocompatible polymers, a process fortheir preparation and compositions containing them.

BACKGROUND

Known in the prior art are polymer derivatives of dextrans obtained bysubstitution by carboxymethyl, carboxymethyl-benzylamide andcarboxymethyl-benzylamide-sulfonate residues. These polymers, theprocess for their preparation and their properties, are described inFrench Patent No. 2,461,724 as well as in U.S. Pat. No. 4,740,594. Amongthese polymers, certain of them imitate the properties of heparin andcan be used as plasma substitution products because of theiranticoagulant and anticomplement properties. Others imitate a differentproperty of heparin which consists of a stabilization, protection andpotentiation of the in vitro biological activity of the growth factorsof the FGF family (Tardieu et al., Journal of Cellular Physiology, 1992,150, pages 194 to 203). Furthermore, French Patent No. 2,644,066describes the use of carboxymethyl-benzylamide-sulfonate derivatives ofdextran, referred to as CMDBS, alone or associated with FGFs, forcicatrization.

More recently, French Patent Nos. 2,718,023, 2,718,024 and 2,718,026proposed the use of polymers capable of protecting, stabilizing andpotentiating growth factors that have an affinity for heparin, such asthe fibroblast growth factors or FGF and Transforming Growth Factor beta(TGFβ), as a drug for the treatment of lesions of the gastrointestinaltract, nervous system and muscle tissues, respectively. To illustratethis protective effect of the CMBDS dextran derivatives, these patentspresent the results of the proteolytic digestion by trypsin of FGF1,FGF2 or TGFβ. These properties of protection, stabilization andpotentiation of the growth factors with an affinity for heparin enabledcharacterization of a new class of polymers, designated as HBGFPP toindicate “Heparin-Binding Growth Factor Protector and Potentiator”, thatexhibit cicatrizing and repair activities in relation to muscle, nervousand gastrointestinal tract tissues, and which are devoid ofanticoagulant activity at the doses employed.

In addition to the above HBGFPP applications, French Patent No.2,718,025 proposes the use of these polymers as drugs for the treatmentof inflammations. This anti-inflammatory activity is illustrated by thein vitro inhibitory action against proteolytic enzymes implicated in theinflammatory reaction such as leukocyte elastase or plasmin and in vivoby histological studies demonstrating a reduced inflammatory cellularreaction in tissues treated by CMDBS dextran derivatives.

However, these CMDBS dextran derivatives are compounds which aredifficult to synthesize and present the risk of salting out residuesthat are known for their toxic effects such as benzylamine. Furthermore,the applications of the HBGFPP and thus of the CMDBS proposed in theprior art are limited solely to the repair and cicatrization of certaintissues.

SUMMARY

The invention relates to a biocompatible polymer constituted by asequence of identical or different components of the general formula(I): A_(a)X_(x)Y_(y), in which A represents a monomer, X represents acarboyxl group fixed on a monomer A, Y represents a sulfate or sulfonategroup fixed on a monomer A; a represents the number of monomers A, xrepresents the substitution rate of the set of monomers A by the groupsX, y represents the substitution rate of the set of monomers A by thegroups Y. The invention also relates to the pharmaceutical or diagnosticcompositions containing at least one polymer of general formula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows formulas of selected β-malic acids.

FIG. 2 shows selected structures of three β-lactones.

FIG. 3 shows the synthesis of alkyl malolactonate from DL-aspartic acid.

FIG. 4 shows the synthesis of derivatives of poly(β-malic acid).

FIG. 5 shows structures of selected monomers A and monomers type A-X,A-Y and A-Z.

FIG. 6 is a table representing selected polymers of type CM_(n)DS_(m)and the percentages of three groups X, Y and Z.

FIG. 7 shows the structure of CM₂DPhSS₁.

FIG. 8 shows the structure of CM₂DES₁.

FIG. 9 shows the structures of CM₃DPheS₂ and CM₃DTyrS₂.

FIG. 10 shows the structures of CM₁DPalmS₁ and CM₁DOleicS₁.

FIG. 11 is a table showing anti-coagulant activities of selectedpolymers.

FIG. 12 is a table showing the stabilizing effects of selected polymerson FGF₁.

FIG. 13 is a table showing the potentiation effects of FGF₁ and FGF₂ forselected polymers.

FIG. 14 is a table showing the percentage of FGF₁ and FGF₂ and TGFβ notdegraded by trypsin in the presence of poly(β-malic acid) polymers.

FIG. 15 is a table showing the percentage of FGF₂ and TGβ not degradedby trypsin in the presence of polymers of the invention derived fromdextrans.

FIG. 16 is a table showing the inhibitory effects of selected polymersof the invention on the activities of leukocyte elastase and plasmin.

FIG. 17 is a table showing percentages of muscular regeneration afterinjection of various doses of polymers.

FIG. 18 is a table showing modulation of the in vitro activity of SOD byselected polymers of the invention.

FIG. 19 is a table showing the protective effect of selected polymers ofthe invention on SOD after treatment by trypsin and thermal shock.

FIG. 20 is a graph showing the potentiation effect of SOD produced invitro by activated monosites.

FIG. 21 is a table showing the inhibitory effects of selected polymersof the invention on calpaine.

FIG. 22 is a table showing the inhibitory effects of selected polymersof the invention on heparitinase.

FIG. 23 is a graph showing the actions of selected polymers of theinvention on the secretion of cologens in vitro by HISM cells subjectedto ionizing radiation of ⁶⁰Co.

FIG. 24 is a graph showing the action of selected polymers of theinvention on the synthesis of Type I, II and V collagens by HISM cellssubjected to ionizing radiation of ⁶⁰Co.

FIG. 25 is a graph showing the protective effects of selected polymersof the invention on the survival of cells subjected to ⁶⁰Co irradiation.

FIG. 26 is a table showing the antifibrotic action of selected polymersof the invention on pig aorta smooth muscle cells.

FIG. 27 is a series of photographs showing the effects of polymers ofthe invention on cutaneous cicatrization.

FIG. 28 is a pair of photographs of the protective effects of polymerRGTA 1005 against tissue injury in a muscle ischemia model in a rat.

FIG. 29 is a series of photographs showing the effects of a polymer ofthe invention RGTA 1015 on the regeneration of long bones andhistological and radiographic studies of femurs from rats which weretreated or not treated.

FIG. 30 is a graph showing the effects of selected polymers of theinvention on the regulation of the osseous mass and on the quality ofits restructuring in chronic periodontal disease.

DETAILED DESCRIPTION

This invention resolves the drawbacks of the prior art by providing newbiocompatible polymers which are easy to prepare and present theproperties of the HBGFPP but also, in an unexpected manner, novelproperties which enable very extensive fields of application, especiallyin therapeutics, which are not limited to just a few types of particularorgans, tissues or cells. The polymers of the invention are, therefore,referred to below as “RGTA” for “ReGeneraTing Agents”.

The polymers of the invention have a molar mass greater than about 5,000da and are constituted by a sequence of identical or differentcomponents of the following general formula (I):A_(a)X_(x)Y_(y)in which:

-   -   A represents a monomer,    -   X represents a carboxyl group fixed on a monomer A and contained        within a group according to the following formula: —R—COO—R′, in        which R is a bond or an aliphatic hydrocarbon chain, possibly        branched and/or unsaturated, and which can contain one or more        aromatic rings, with the exception of benzylamine and        benzylamine sulfonate, and R′ represents a hydrogen atom or a        cation,    -   Y represents a sulfate or sulfonate group fixed on a monomer A        and contained within a group according to one of the following        formulas: —R—O—SO₃—R′, —R—N—SO₃—R′, —R—SO₃—R′, in which R is a        bond or an aliphatic hydrocarbon chain, possibly branched and/or        unsaturated, and which can contain one or more aromatic rings,        with the exception of benzylamine and benzylamine sulfonate, and        R′ represents a hydrogen atom or a cation,    -   a represents the number of monomers A, which is such that the        mass of the polymers of formula (I) is greater than        approximately 5,000 da,    -   x represents the substitution rate of the set of monomers A by        the groups X, which is comprised between approximately 20 and        150%, preferably on the order of 50%,    -   y represents the substitution rate of the set of monomers A by        the groups Y, which is comprised between approximately 30 and        150%, preferably on the order of 100%.

The radical R of the groups X and Y in formula (I) is advantageouslyselected from among an alkyl, allyl, aryl, linear or branched group.

In the definition of the substitution rates cited above, a substitutionrate x of 100% is understood to mean that each monomer A of the polymerof the invention contains statistically one X group. Similarly, asubstitution rate y of 100% is understood to mean that each monomer ofthe polymer of the invention contains statistically one Y group.Substitution rates higher than 100% manifest the fact that each monomerbears statistically more than one group of the type under consideration.Conversely, substitution rates lower than 100% manifest the fact thateach monomer bears statistically less than one group of the type underconsideration.

The polymers offer the remarkable advantage of presenting in vivo a slowdegradability, which differentiates them from the heparan sulfates whichare products that are naturally and rapidly degraded by heparinase orheparitinase. Furthermore, the polymers of the invention neither containnor release toxic products after degradation, in contrast to the CMDBS,which have benzylamine groups.

The monomers A which constitute the base elements of the polymers offormula I can be identical or different, and are selected from among alltypes of monomers such as, for example, those proposed for the HBGFPPsuch as the sugars, esters, alcohols, amino acids, nucleotides and thelike. Among these, preference is given especially to the -oses, andespecially to glucose.

The number of monomers A is defined in a manner such that the mass ofthe polymers of the invention is greater than approximately 5,000 da.Consequently, the RGTA can be constituted by diverse homomeric as wellas heteromeric polymerizable structures such as, for example: thepolyester copolymers of biosynthesis or chemical synthesis such as thealiphatic polyesters or those of natural origin such as thepolyhydroxylalcanoates. Also applicable are polysaccharides and theirderivatives of bacterial origin such as cellulose, xanthan, dextran andthe like, unicellular or multicellular plant extracts such as starch,plant cellulose or alginates or their derivatives, the fucanes and theirderivatives and the like, or animal extracts such as hyaluronic acid orchitin and the like, or products of synthesis such as those obtained bycopolymerization. Synthesized proteins such as the natural or chemicallymodified polyamino acids such as, for example, collagen can also beemployed in the constitution of the polymers of the invention.

The selection of the polymer structure of the RGTA is based on thevarious forms of presentation which can be used depending on the tissueor the organ to be treated or the physicochemical characteristics suchas the solubility, the spatial conformation with the solutions,suspensions, gels, powders, sponges, films, compact moldable materials,porous, semiporous or nonporous materials, materials that crumble ormaterials that do not crumble, materials that erode and materials thatdo not erode, materials that can be colonized or materials that can notbe colonized, etc.

Heparin or its fragments with weak anticoagulant properties and naturalheparan sulfates and their fragments are excluded from the scope of thepresent invention because:

-   -   Their anticoagulant activity can be greater than 50 IU. In fact,        the polymers of the invention are devoid of significant        anticoagulant activity, i.e., they present an activity lower        than about 50 IU/milligram compared to that of heparin whose        activity is on the order of 150 to 170 IU/milligram.    -   They exhibit biodegradability under the action of enzymes of the        heparinase or heparitinase type which is too rapid to enable the        biological effects obtained in the context of this invention.

The polymers of the invention can also comprise functional chemicalgroups, designated Z, which are different from X and Y, and capable ofconferring on the polymers of the invention supplementary biological orphysicochemical properties. Thus, the groups Z are useful for conferringon the RGTA a better solubility or lipophilic properties enabling betterdiffusion or tissue penetration, for example, increasing the amphiphilicproperties, enabling crossing the blood-brain barrier and the like. Asan example, the groups Z, which can be identical or different, can beamino acids, fatty acids, fatty alcohols, ceramides or derivativesthereof, or nucleotide addressing sequences.

The groups Z can also represent identical or different active agents,such as therapeutic or diagnostic agents, for example, ananti-inflammatory, antimicrobial, antibiotic and the like or an enzyme,a growth factor and the like.

The polymers of the invention in which Z is present have the followingformula II:A_(a)X_(x)Y_(y)Z_(z)in which A, X, Y, a, x, and y have the same meanings as defined aboveand z represents the substitution rate of the set of monomers A by thegroups Z, which is comprised between approximately 0 and 50%, preferablyon the order of 30%. The groups X, Y and Z can be fixed directly on themonomer A or fixed to each other with only one of them being fixed tothe monomer A. Thus, the groups Z can be fixed by covalence directly onthe monomers A or fixed by covalence on the groups X and/or Y.

However, the groups Z can also be conjugated to the polymers of formula(I) by bonds other than covalent bonds, such as ionic or hydrophilicinteractions, depending on the nature of A, X and Y. The polymers of theinvention can then constitute a vectorization system of Z. Thus, thepolymers of the invention are useful for transporting an active agent Z,such as a growth factor or an enzyme, to the level of the injured ordiseased tissue as defined below in the context of the applications ofthe polymers of the invention.

The polymers in which the groups R of X and Y or the groups Z arecapable of inducing toxic effects directly or after degradation are notincluded within the scope of this invention. Among the groups Z excludedfrom the invention can be cited those groups constituting mutagenicagents or those known to be carcinogenic or exhibiting other toxicproperties. This is the case of benzylamine which can be precipitatedout from the benzylamide group contained in the CMDBS and thecarcinogenic properties of which are well known by the expert in thefield (Wiessler M, et al., Carcinogenesis 1983; 4(7): 867-871; Singer GM, et al., J Med. Chem. 1983; 26(3): 309-312). CMDBS is consequentlyexcluded from the polymers of the invention.

The invention also pertains to the pharmaceutical or diagnosticcompositions containing at least one polymer as defined above associatedin the composition with a pharmaceutically acceptable vehicle. Thesecompositions can be used in humans as well as in animals. In fact, aswith the HBGFPP described in the prior art, the polymers of theinvention present the following properties:

-   -   They are devoid of significant anticoagulant activity, which        means that they present an activity lower than about 50        IU/milligram compared to that of heparin the activity of which        is on the order of 150 to 170 IU/milligram.    -   They stabilize and potentiate the growth factors that exhibit an        affinity for heparin and particularly, as an example, FGF1        and/or FGF2 and TGFβ.    -   They protect these factors against proteolytic agents such as        trypsin.    -   They inhibit the protease activities implicated in the        inflammatory process such as, for example, leukocyte elastase or        plasmin.    -   They exert a cicatrizing effect in at least the models presented        in the cited patents on muscles, nerves or the gastrointestinal        tract.

Thus, the polymers of the invention constitute a new class of agentsthat promote the repair of muscle and nervous tissues, and those of thegastrointestinal tract and which present, as reported in U.S. Pat. No.5,693,625 for the CMDBS, properties on cutaneous cicatrization and thatof the cornea or on flat bone as described by Blanquaert F et al. (Bone,1995; 17(6): 499-506). However, the polymers of the invention alsopresent unexpected properties in relation to the CMDBS. As an example,we can cite the effect of the CMDBS on cranial bone defects (BlanquaertF et al., Bone, 1995; 17(6): 499-506) which a priori is not transposableto long bone. Flat bone and long bone are of a completely differentnature because:

-   -   They are of different embryological origin. Flat bone is a        derivative of the conjunctive tissue cells whereas long bone is        a derivative of the cartilaginous cells.    -   Flat bone is a spongy bone and does not contain a medullary        cavity in contrast to long bone.    -   Flat bone does not cicatrize naturally when there is a loss of        substance such as upon trepanation as can be seen by the absence        of cicatrization in the crania originating from prehistoric        civilizations such as the Incas or Egyptians (see for example        Evidence for stone age cranial surgery, 7000 BC, Nature, 1997,        367, 360). Surprisingly, the polymers of the invention present        effects on the repair of very severe bone defects on the order        of a third of the length of the diaphysis of a rat femur. Not        only was repair observed with reformation of an osseous shaft        but also the shaft was filled with marrow, i,e., the treatment        with RGTA led to the reformation of a true medullary cavity        whereas in the absence of RGTA the defect is simply packed with        disorganized osseous material. The same is true of cutaneous        incisions because this repair is of such high quality that there        is practically no trace of a scar.

The invention, therefore, pertains more particularly to the previouslydescribed biocompatible polymers of formulas (I) and (II) which aredevoid of significant anticoagulant activity. These polymers present ananticoagulant activity lower than about 10 IU/mg at single or dailydoses comprised:

-   -   between about 0.001 mg and about 1 mg per cm³ when the        application is local,    -   between about 0.1 mg/kg and about 100 mg/kg when the        administration is via the systemic route, for example, via the        intravenous route,    -   between about 0.2 mg/kg and about 500 mg/kg when the        administration is via the intramuscular route,    -   between about 0.1 mg/kg to about 5 mg/kg when administered        orally.

The anticoagulant activity of the polymers of the invention at the dosesindicated above is for a person weighing about 70-kg lower than:

-   -   about 10 IU for a local application,    -   about 100 IU for an intravenous administration,    -   about 500 IU for an intramuscular administration.

Remarkably, the polymers of the invention are devoid of significantanticoagulant activity at the doses specified above but still presentthe capacity of preserving or restoring tissue homeostasis. Theinvention, therefore, pertains particularly to the use of theaforementioned polymers for the preparation of a drug that is useful forthe prevention or treatment of dysfunctions of tissue homeostasis andwhich do not present significant anticoagulant activity.

In a completely surprising manner, we found that the polymers of theinvention can be administered via routes other than those described inthe prior art for the CMDBS which only envisaged local administration atthe level of the injured tissue. Thus, it is possible to select amongthe polymers of the invention those which are suitable, for example,because of their solubility, for the selected route of administration:intravenous, intra-arterial, intramuscular, intraperitoneal,intraocular, into the cerebrospinal fluid or directly into the centralnervous system to cross over the blood brain barrier, as well as orally.All of these routes of administration, possibly even combined, have beenshown to be particularly effective.

Thus, the invention concerns pharmaceutical compositions in which thepolymer of the invention is associated with a pharmaceutical vehicle ina form enabling oral, cutaneous, subcutaneous, topical, intramuscular,intravenous or intra-arterial administration or administration into anyof the fluid compartments of the organism. Studies performed in theframework of this invention revealed that the RGTA act especially withinthe dosage windows beyond which satisfactory biological effects were notalways obtained. These optimal effective doses are defined below anddepend on the route of administration and the type of lesion.

Advantageously, in a manner to completely cover the surface of a woundor to fill the volume of a tissue lesion, the pharmaceuticalcompositions are dosed to allow administration of about 0.001 to about 1milligram of polymer per square centimeter or of about 0.005 to about 1am of polymer per cubic centimeter of tissue to be treated. Thus, thecompositions of the invention preferably contain between about 0.01 andabout 10 milligrams of polymer per milliliter of physiological solutionof dissolution.

For administration via the systemic route (venous, arterial), via theintraperitoneal route or intraocular route, into the cerebrospinalfluid, into the intracochlear fluid or into any peritissular orintratissular fluids, the compositions of the invention are dosed toallow administration of between about 0.1 and about 100 milligrams ofpolymer per kilogram of weight of the human or animal to be treated.

For administration via the intramuscular route, the compositions of theinvention are dosed to allow administration of between about 0.2 andabout 500 milligrams of polymer per kilogram of weight of the human oranimal to be treated, preferably between about 1 and about 50 mg per kg.

For oral administration, the compositions of the invention are dosed toallow administration of between about 1 and about 5000 milligrams ofpolymer per kilogram of weight of the human or animal to be treated,preferably between about 10 and about 500 mg per kg.

Furthermore, the previously reported properties of the HBGFPP, i.e.,that they present a cicatrizing and repairing activity on the muscle andnervous tissues and on those of the gastrointestinal tract and that theycan be used as drugs for the treatment of inflammations, we havedemonstrated the remarkable effects of the polymers of the invention onthe regulation of the homeostasis of the mass and the functionality ofthe tissues and organs, demonstrating that they act on tissular andcellular regeneration, protection, preservation and aging in vivo and exvivo, as well as antifibrotic activities and protective activitiesagainst the deleterious effects of ischemias, ionizing radiation andoxidizing products induced by diseases or stress or stemming from food.Consequently, the RGTA should be considered to be regulators of tissularhomeostasis in that they regulate the mass of the regenerated tissuesand they act on the reorganization of the matrix in that they exert anantifibrotic and cicatricial effect on the tissues and organs subjectedto acute as well as chronic destructive processes.

These properties of the RGTA are characterized by unique effects on thequality and rate of the tissue repairs. These effects are manifested byan almost complete reconstitution and identity with the original tissuestructure (prior to the lesion) and by the almost complete absence ofcicatricial traces such as in particular signs of fibrosis or loss ofits functional integrity. More particularly, the research results whichwill be presented below in the experimental part demonstrated the invivo tissue protection and regeneration properties of the RGTA in thefollowing models:

-   -   cutaneous wound lesion,    -   regeneration of osseous tissues such as long bones with the        example of the femur,    -   protection against the loss of osseous tissue and regulation of        its restoration in a model of chronic periodontal disease,    -   protection against the deleterious effects of ionizing        radiation,    -   protection against the deleterious effects of ischemias        irrespective of their location.

Thus, the properties below of the polymers of the invention have beenparticularly demonstrated:

-   -   A protective effect against the deleterious effects linked to        oxidative stress and to oxidizing agents. The polymers of the        invention act in particular as protective agents and        potentiators of enzymes such as superoxide dismutase or SOD.        This property makes it possible to use the polymers of the        invention for the preparation of a drug intended for the        treatment and/or the prevention of lesions and disorders induced        by oxidative stress and oxidizing agents. But this property also        makes it possible to use the polymers of the invention alone or        in association with another compound as a preservative for foods        or nutriments that naturally contain antioxidants or to which        antioxidants, such as animal or plant SOD, were added.    -   A preservative and protective effect against ischemias. This        property makes it possible to use the polymers of the invention        for the preparation of a drug intended for the treatment and/or        the prevention of pathologies associated with hypoxia, with        cellular degeneration such as the neuropathies, myopathies,        hepatopathies, nephropathies, the cardiopathies and the like and        to the peroxidations of molecules such as the lipids. This        property also makes it possible to use the polymers of the        invention in compositions for the preservation of the        functionality of tissues and organs especially for the purpose        of their conservation, the transport of ex vivo organs, organ        transplants, their grafting as well as prostheses.    -   An inhibitory activity against the enzymes of the calpaine        family. This property makes it possible to use the polymers of        the invention for the preparation of a drug intended for the        treatment and/or prevention of heart diseases and diseases of        the nervous system.    -   An inhibitory activity against the degradation enzymes of        heparin or the heparan sulfates such as heparinase or        heparitinase. This property makes it possible to use the        polymers of the invention for the preparation of a drug intended        for the treatment and/or the prevention of the diseases        associated with an anarchic growth of the cells and the        pathological processes in relation with an angiogenesis.    -   An action of protecting cells against the effects of ionizing        radiation and of both the quantitative and qualitative        regulation of the constituents of the cell matrix such as, for        example, the collagens, making it possible to increase the        survival and functioning of the cells. This activity makes it        possible to use the polymers of the invention for the        preparation of a drug intended for the treatment and/or        prevention of the deleterious effects of ionizing radiation as        well as the use of these polymers in the preparation of cosmetic        products.    -   An antifibrotic activity which is manifested in vitro and in        vivo by regulatory effects on the proliferation of mesenchymal        cells such as the smooth muscle cells, fibroblasts or hepatic        cells and the quality of the type of collagen that they secrete,        as well as an activity on the phenotypic quality of the        collagens synthesized by these cells and by the notable        reduction in the fibrotic cicatricial sequelae. This activity        makes it possible to use the polymers of the invention for the        preparation of a drug intended for the treatment and/or        prevention of pulmonary, renal, hepatic, cardiac, vascular and        dermatological pathologies as well as pathologies of grafts and        their functional integration, of multiple derivatives linked to        parasitism.

Thus, unexpectedly, the inventors discovered that the RGTA presented thecapacity to:

-   -   protect, stabilize the enzymatic activities of superoxide        dismutase or SOD,    -   potentiate the enzymatic activities of superoxide dismutase or        SOD.        Thus, this enzyme can be protected either in situ and in vivo or        ex vivo. The RGTA can, therefore, be used alone as protective        agents of endogenous SOD and in this role protect SOD in all of        its functions or it can be used in association with SOD in the        indications known by those of ordinary skill in the therapeutic        and cosmetic fields. Due to this property, the RGTA also act as        preservatives of foods or nutrients or nutriments.

The RGTA act as protective and reparative agents of the deleteriouseffects associated with tissue stress. Thus, in ischemia, regardless ofits origin or in response to a cellular or tissular aggression, forexample, under the effect of ionizing radiation, or in response to aninvasion by a pathogenic agent, regardless of whether it be viral,microbial, parasitic or even of the type causing pathologies of the TSSE(Transmissible Subacute Spongiform Encephalopathies) type such asprions, or during vascular rupture caused by hemorrhages whose effectsare harmful by causing functional losses, for example, in the case ofhemorrhages of the retinal vessels which can lead to blindness or in thebrain which can lead to loss of motor functions or others.

Consequently, the invention pertains to pharmaceutical compositionscontaining at least one RGTA in both human and animal fields ofapplications. These compositions are beneficial in medical, veterinaryand cosmetic fields as well as in alimentary fields as preservatives offoods or nutriments that naturally contain antioxidants or to which areadded antioxidants such as animal or plant SOD.

The protective qualities of endogenous SOD or SOD that is providedexogenously are reinforced by the RGTA. Thus, the RGTA can beadministered in all cases in which the beneficial effects of SOD havebeen described.

For the treatment of diseases in which exogenously supplied SOD has beenshown to be effective, the therapeutic effects resulting from theprotective effect of endogenous SOD can then act more effectively in thepresence of RGTA. The RGTA limit or avoid exogenous intakes from actingindirectly as antioxidant agents. The following therapeutic effectsresult from these effects:

-   -   Protection against the aggressions of disease or degeneration of        the nervous tissues. For example in stress (Shahen et al.,        Effects of various stressors on the level of lipid peroxide        antioxidants and Na⁺, KT⁺—ATPasc activity in brain, Experentia,        1996, 52, 336-339) or of neuronal degeneration and the        neurodegenerative diseases associated with vascular accidents,        traumatic accidents or of pathologies such as Parkinsonism in        which the protective activity of endogenous SOD enables a more        intensive effect (Bostantjopoulos et al., Superoxide dismutase        activity in early and advanced Parkinson's disease, Funct.        Neurol., 1997, 12, 63-68).    -   Aging due to an antiapoptosis effect (Fernandez Novoa et al.,        Methods Find Exper Clin Pharmacol, 1997, 9, 99-106).    -   An antioxidant protection in ischemias of the limbs. The RGTA        can be administered by itself in this application. The        protective provided by treatment with SOD alone will be        reinforced by the administration of RGTA. In view of the        properties described, that the RGTA is beneficial when        administered alone and/or in association with SOD in the        numerous applications describing the effects of SOD (D'Agnillo        and Chang, Reduction of hydroxyl radical generation in a rat        hind limb model of ischemia—reperfusion injury using        cross-linked hemoglobin—superoxide dismutase—catalase, Anif.        Cells Blood Substiti Immobil Biotechnol, 1997, 25, 163-80).

The administration of RGTA alone or in association with. SOD would bebeneficial against disorders of the heart, brain and central nervoussystem as in the case of lesions of the spinal cord (Nakauchi et al.,Effects of lecithinized superoxide dismutase on rat spinal cord injury,J. Neurotrauma, 1996, 13, 573-82) or of the retina.

In the treatment of respiratory insufficiencies associated withdiaphragmatic fatigue (Supinski et al., Effects of free radicalscavengers on diaphragmatic fatigue, Am. J. Respir. Crit. Care Med.,1997, 155, 622). This could be notably of value in patients withmuscular dystrophy.

All tissues, especially those for which the endogenous active SOD levelis higher (vascular endothelial cells, in the liver, the kidneys, thecardiac and skeletal muscles, the pancreas, the epithelial cells of theintestinal mucosa, the colon, the trachea, the esophagus, theconjunctive tissue and cartilage).

The treatment of the deleterious effects associated with diabetes suchas the diabetic retinopathies (Szabo et al., Direct measurement of freeradicals in ischemic/reperfused diabetic rat retina, Clin. Neurosci.,1997, 4, 240-5).

The treatment of leprosy (Serum, zinc, copper, magnesium, proteins andsuperoxide dismutase in leprosy patients on multidrug therapy—afollow-up study, Indian J. Lepr., 1996, 68, 325-53).

In the treatment of endotoxic shock (Hepatic response to the oxidativestress induced by E. coli endotoxin, Mol. Cell. Biochem. 1996, 159,115-121).

In the treatment of the lesions induced by the stress caused by thepresence of pathogenic agents of all types, especially viruses,including the AIDS virus, or the agents that induce TSSE such as theprions.

In the preventive and/or curative treatment against irradiations. Thus,the adverse effects of radiotherapy can be reduced by a preventiveand/or curative treatment with RGTA. The use of the RGTA allowsreduction of the dose of radiotherapy under cancer treatment conditions(Prevention of radioinduced cystisi by orgotein; a randomized study,Anticancer Res., 1996, 16, 2025-8) or enable prevention of theclastogenic effects induced by the irradiation. The hyperthermic effectsassociated with radiotherapy can be diminished by the use of RGTA in thesame manner as SOD (Kandasamy et al., Involvement of superoxidedismutase and glutathione peroxidase in attenuation of radiation-inducedhyperthermia by interleukin 1 alpha in rats, Brain Res., 1993, 606,106-10).

In the protection against the effects induced by ultraviolet radiation,for example on the retina. (Oguni et al., Chronic retinal effects byultraviolet irradiation with special reference to superoxide dismutase,Histol. Histopathol., 1996, 11, 695-702) and the treatment of uveitis(Koch et al., Effects of different antioxidants on lens-induced uveitis,Ger. J. Ophthalmol., 1996, 5; 1185-8). The treatment with RGTA can bealone or in association with SOD and its use can be medical as well ascosmetic.

In the treatment of hypertension. In fact, the activity of endogenousSOD is diminished in subjects with hypertension (Jun Ke Yan andCatalano, Increased superoxide anion production in humans, a possiblemechanism for the pathogenesis of hypertension, J. Hum. Hypertens. 1996,10, 305-309). The intake of RGTA can have the effect of augmenting thisactivity and reducing the hypertension.

-   -   In the treatment of inflammatory diseases such as arthritis.

The RGTA are also useful for the conservation of organs or tissues aswell as in the maintenance of biological fluids such that the followingapplications can be envisaged:

-   -   In the biological fluids such as blood and its cellular        constituents, for example, in hemodialysis.    -   For the preservation of organs in the case of grafts or ex vivo        treatment or in reperfusion (Razak et al., Cross-linked        hemoglobin-superoxide dismutase-catalase scavenges free radicals        in a rat model of intestinal ischemia-reperfusion injury, Artif.        Cells Blood Substiti Immobil. Biotechnol. 1997, 25, 181-192).        The addition of RGTA to solutions for organ conservation and        those administered in reperfusion enables preservation of these        organs.

The invention pertains to pharmaceutical compositions containing atleast one polymer as defined above and intended for the treatment and/orprevention of tissular lesions and disorders, such as those found intraumatology, requiring reparative or plastic surgery of cutaneous ordeeper floors such as those of muscles, bone, brain, heart, viscera,etc., in the degenerative diseases possibly associated with fibrosis, inthe losses of tissue mass such as osteoporosis or myopathies, in thecardiovascular and neurological fields, in dermatology, etc.

A remarkable property of the RGTA is their capacity to fix themselves onthe injured tissues, which enables the use of the RGTA as targetingvectors in a therapeutic and/or diagnostic objective. Consequently, thepolymers of the invention can be coupled to molecules, represented aboveby the group Z, endowed with therapeutic activities or to molecules thatfacilitate the repair of the injured tissue.

Other advantages and characteristics of the invention will becomemanifest from the examples below concerning, on the one hand, thepreparation of polymers of the invention in which the polymer skeletonis of the polyester type or of a polysaccharide nature and, on the otherhand, the properties of the polymers of the invention.

A) Preparation of the Polymers of the Invention

EXAMPLE 1 Synthesis of poly(β-malic acid) Derivatives from aNon-polysaccharide Skeleton

In this example, the polymer of the invention is a copolymer of β-malicacids of general formula (II), the components A of which, substituted byX and/or Y and/or Z, are represented in FIG. 1. In FIG. 1:

-   -   A is —(O—CH₂—CH₂—CO)—    -   X is —COOH or —COO⁻Na⁺    -   Y is —CO—CH₂—CHOH—CH₂—SO₃H or —CO—CH₂—CHOH—CH₂—SO³⁻Na⁺    -   Z is —CO—OCH₃—CH(CH₂—CH₃)—CH₃    -   x, y and z correspond to the percentages of the X, Y and Z        groups shown in Table I below in relation to the different        polymers synthesized.

TABLE I Type of Carboxylic Sulfonate Hydrophobic Reference polymergroups = X groups = Y groups = Z RGTA 2010 Pcoo⁻ 100%  0%  0% RGTA 2011P1S  60% 10% 10% RGTA 2012 P2S  75% 11% 12%

Pcoo⁻ corresponds to a polymer composed exclusively of carboxylic orcarboxylate groups X. P1S and P2S correspond to polymers composed ofsulfonate groups Y and hydrophobic butyl groups Z in addition to thegroups X.

The synthesis of this polymer proceeds from the preparation ofβ-substituted β-lactones and is followed by an anionic polymerization byring opening. The three β-lactones synthesized are shown in FIG. 2.

The monomers are obtained from DL-aspartic acid in four steps prior tothe polymerization as shown in FIG. 3.

The synthesis of the alkyl malolactonate is performed from DL-asparticacid in which R represents —CH₂—C₆H₅ (benzyl malolactonate) or—CH(CH₃)—CH₂—CH₃ (2-butyl malolactonate) or CH₂—CH═CH₂ (allylmalolactonate).

I—Synthesis of the Monomers (Aspartic Acid Pathway)

1) Synthesis of (2R,S)bromosuccinic acid

Synthesis of (2R,S)bromosuccinic acid is obtained after a diazotationreaction of DL-aspartic acid. For this reaction, the carbon bearing theamine group undergoes a double inversion of configuration (Guérin etal., Optically active poly(β-malic acid), 1985, Polymer Bulletin, 14:187).

100 g (0.75 moles) of DL-aspartic acid and 415 g (4.04 moles; 5.5 eq.)of sodium bromide are dissolved in 1620 milliliters of 2N sulfuric acidin an ice bath. Then 62 g (0.9 moles; 1.2 eq.) of sodium nitrite areadded in small quantities, under agitation. Thirty minutes after the endof this addition, the reactional medium is neutralized with 8 g (0.13moles; 0.18 eq.) of urea for 30 minutes at room temperature. Thebromosuccinic acid is extracted with 1 liter of ethyl acetate and theaqueous phase washed with 1 liter of ethyl acetate. The organic phasesare dried over magnesium sulfate, filtered on a Büchner funnel then theethyl acetate is eliminated in the Rotavapor.

The bromosuccinic acid is then recrystallized 4 times in acetonitrile,filtered on a Büchner funnel then dried for 4 h at 45° C. under vacuumin a desiccator.

-   -   Characteristics: MW=197; m=52.8 g; Yield of 36%; mp=168° C.;        appearance: white powder.        2) Synthesis of the Monoesters

The synthesis of the monoesters comprises the prior preparation of theanhydride without racemization of the chiral centers. The anhydride issynthesized from (2R,S)2-bromosuccinic acid under the action of adehydrating agent, trifluoroacetic anhydride (TFAA), under anhydrousconditions and under nitrogen. The following step is the opening of theanhydride by an alcohol which leads to two monoesters, only one of whichwill be lactonizable. The choice of the alcohol is based on the natureof the desired lactone.

a) Synthesis of benzyl bromosuccinate

Fifty g (0.25 moles) of bromosuccinic acid are degassed under a nitrogenstream for 2 h. In an ice bath, 125 milliliters of anhydroustetrahydrofuran (THF) then 43.4 milliliters (0.30 moles; 1.2 eq.) oftrifluoroacetic anhydride (TFAA) are added drop by drop by a droppingfunnel. The solution is left for 2 h at room temperature underagitation, then the THF, the TFA formed and the TFAA in excess areeliminated in the Rotavapor. The bromosuccinic anhydride is allowed todegas for 2 hours.

Then 27.7 milliliters (0.25 moles; 1 eq.) of benzyl alcohol are added.The solution is agitated at 40° C. under an inert atmosphere for 12 h.The mixture of monoesters obtained in this manner is dissolved in 150milliliters of ether. The etherized phase is washed 3 times with 100milliliters of water, dried on magnesium sulfate and filtered on aBüchner funnel.

-   -   Characteristics: MW=287; m=71.3 g; Yield of 95%; appearance:        pale yellow oil.        b) Synthesis of allyl bromosuccinate

The same protocol as previously employed is used but 17.86 milliliters(0.25 moles; 1 eq.) of allyl alcohol are added. The reactional medium isagitated for 22 h at 60° C. under an inert atmosphere rather than for 12h at 40° C. The monoesters are purified in identical manner.

-   -   Characteristics: MW=237; m=17.6 g; Yield of 72.8%; appearance:        viscous oil.        c) Synthesis of 2-butyl bromosuccinate

The same protocol as above in 2)b), but with 18.8 g (1 eq.) of(RS)2-butanol that has been distilled in advance. The reactional mediumis agitated for 12 hours at 60° C. under an inert atmosphere.

-   -   Characteristics: MW=253; m=45 g; Yield of 90%; appearance:        orangish yellow oil.        3) Synthesis of the Lactones

The lactonization reaction is performed on the sole lactonizablemonoester and presents an inversion of configuration. It is performeddirectly on the mixture of monoesters after neutralization at pH 7.2with 2N soda. The reaction takes place in a dichloromethane/waterbiphasic medium. The lactone is purified by silica columnchromatography. The nature of the eluent varies depending on the natureof the lactone. This lactone is then distilled on an appropriate column.

a) Synthesis of benzyl malolactonate

71 g (of which 70% is lactonizable monoester, i.e., 0.173 moles) of themixture of monoesters are dissolved in 300 milliliters of ether and 250milliliters of water in a balloon flask. A 2N solution of sodiumhydroxide is added drop by drop until reaching pH 7.2, then 450milliliters of dichloromethane are added. The flask is placed on arefrigerant system and the biphasic system is strongly agitated for 3 hat 40° C.

After decantation, the organic phase is washed 2 times with 250milliliters of water then 2 times with 250 milliliters of brine, driedover magnesium sulfate and filtered. The solvent is eliminated in theRotavapor. This lactone is purified on silica column (eluent: 8/2dichloromethane/petroleum ether) and distilled 3 times under vacuum.

-   -   Characteristics: MW=206; m=20.7 g prior to purification, i.e. a        yield of 40.5%; m=3.41 g after purification, i.e., a yield of        6.7%; bp=116-118° C. under 3·10⁻² mbar; IR (n, cm⁻¹): ^(n)(CO        lactone)=1825 cm'⁻¹; ^(n)(CO ester)=1740 cm⁻¹; appearance:        colorless oil.        b) Synthesis of allyl malolactonate

According to the same protocol as above in 3)a), but the pH of theaqueous phase is 7.8 rather than 7.2 and the solution is agitated for 5h rather than 3 h. The lactone is purified on silica column (eluent: 4/6petroleum ether/ethyl ether) and is distilled 3 times under vacuum.

-   -   Characteristics: MW=156; m=15.3 g prior to purification, i.e., a        yield of 53.4%; m=4 g after purification, i.e., a yield of 13%;        bp=62-65° C. under 3·10⁻² mbar; IR (n, cm⁻¹): ^(n)(CO        lactone)=1825 cm⁻¹; ^(n)(CO ester)=1740 cm⁻¹; appearance:        colorless liquid.        c) Synthesis of 2-butyl malolactonate

The same protocol is employed as in 3)a) above. The lactone is purifiedon silica column (eluent: 8/2 dichloromethane/petroleum ether) and isdistilled 3 times under vacuum.

-   -   Characteristics: MW=172; m=26.7 g prior to purification, i.e., a        yield of 55%; m=14.4 g after purification, i.e., a yield of 28%;        bp=80-82° C. under 3·10⁻² mbar; IR (n, cm⁻¹): ^(n)(CO        lactone)=1825 cm⁻¹; ^(n)(CO ester)=1740 cm⁻¹; appearance:        colorless viscous oil.        II—Synthesis of the Polymers

The lactones were polymerized in the presence of tetramethyl ammoniumbenzoate (10⁻³ eq.) at 37° C. for 15 days. The polymerization wasmonitored by infrared analysis with observation of the disappearance ofthe lactone band at 1850 cm⁻¹.

1) Synthesis of allyl butyl-co-malate benzyl-co-malate polymalate

Five g (24.2 mmoles) of benzyl malolactonate, 1.4 g (9.3 mmoles) ofallyl malolactonate and 0.7 g (4.1 mmoles) of butyl malolactonate aredegassed for 2 h and transferred via cannula into a balloon flaskcontaining 471 milliliters of a primer solution (tetraethylammoniumbenzoate: 10⁻³ eq.; 37.72·10⁻⁶) at 80·10⁻³ M which was degassed inadvance for 2 h under a nitrogen stream. The copolymerization wasperformed at 37° C. for 15 days under an inert atmosphere and underagitation. The polymer was then dissolved in a minimum of chloroform.The chains were terminated by addition of a drop of concentratedhydrochloric acid and the polymer was precipitated with ethanol thendried under vacuum at 40° C. for 48 h.

-   -   Characteristics: m=4.37 g; yield of 61.5%; Tv=−5° C.; IR (n,        cm⁻¹): ^(n)(C═O)=1748 cm⁻¹; SEC (THE, polystyrene standard);        Mn=6600; MW=9200; Ip=1.4; MSEC=10,000; S 134 (CH═); 168 (C═O        lateral chain); 170 (C═O principal chain); appearance:        transparent vitreous polymer.        2) Epoxidation of allyl butyl-co-malate benzyl-co-malate        polymalate

1.12 g (7.91 mmoles of unsaturated units) of polymer are dissolved in 3milliliters of anhydrous dichloromethane in a balloon flask. A solutioncontaining 466.34 milligrams (4.69 mmoles; 6 eq.) ofmetachloroperbenzoic acid (MCPBA) in 2 milliliters of dichloromethane isadded by cannula. The mixture is agitated for 24 hours at roomtemperature. The polymer is then precipitated with ethanol and dried ina desiccator under vacuum.

-   -   Characteristics: m=4 g; precipitation yield of 92%; epoxidation        reaction yield of 100%.

The molar mass did not change upon epoxidation because MCPBA does notinduce a modification of the chain length.

3) Hydrogenolysis of allyl butyl-co-malate benzyl-co-malate polymalate

Four g of the polymer are dissolved in 5 milliliters of freshlydistilled dioxane on sodium in a balloon flask. 800 milligrams (20% byweight) of palladium on active charcoal are added and the hydrogenolysisis begun. When the volume of hydrogen consumed no longer increases (24 hafter the beginning of the reaction), the hydrogenolysis is stopped. Thesolution is filtered on Celite and the dioxane is eliminated in theRotavapor.

-   -   Characteristics: m=2 g; hydrogenolysis yield of 100%.        4) Sulfonation of allyl butyl-co-malate benzyl-co-malate        polymalate

Four g of polymer (i.e., 5.64·10⁻³ moles of epoxide) are dissolved in 20milliliters of water. 2.14 g of sodium bisulfite (2 eq., 11.28·10⁻³moles) are added. The pH of the solution is adjusted to 7.4(Housse-Ferrari, “Preparation and characterization of porous silicasmodified by polymers and copolymers of N,N′-dimethyl acrylamide”,Thesis, University of Paris VI, Jan. 30, 1990). The solution is leftunder agitation for 7 h in ice then ultrafiltered for 24 h at 4° C.against water and lyophilized.

FIG. 4 summarizes the synthesis steps of the poly(β-malic acid)derivatives.

EXAMPLE 2 Synthesis of Polysaccharide Polymers Constituted bySubstituted Glucose Motifs

I—Synthesis of carboxymethyl dextran sulfates Designated CMDS

In this example, the polymer of the invention is constituted ofsubstituted dextran in which the glucose A motifs substituted by Xand/or Y and in which Z is nothing are represented in FIG. 5. Thedifferent types of grafting are shown in FIG. 5 in which:

-   -   A is a glucose monomer on which X, Y and Z are grafted by the        intermediary of the hydroxyl functions in position 2 and/or        position 3 and/or position 4 and/or by the intermediary of the Y        groups for Z,    -   X is —CH₂COOH or —CH₂COO⁻Na⁺    -   Y is —SO₃H or SO₃ ⁻Na⁺    -   Z is a variable group of which several examples are presented        below.

The polymers of type CMDS in which Z=nothing contain multiple types ofmonomers. The first types of substituted monomers are the carboxymethylglucose of type A-X substituted in position 2 and/or 3 and/or 4 (motifspresented in FIG. 5). The addition of the group Y=(motifs A-Yrepresented in FIG. 5) corresponds to an O-sulfation and becomes, withR=nothing and R′=H⁺ or Na⁺, Y=O—SO₃ ⁻H⁺/Na⁺. If Y is fixed on X, R of Ybecomes CH₂—CO and the lost functionality of X (COO⁻) is no longerconsidered to exist. It then becomes part of Y and enters into themeasurement of the percentages of substitution of the active groups Y.

The different monomers constituting the CMOS polymer are thus eitherunsubstituted glucose or glucose carboxymethyl or glucose sulfate orglucose carboxymethyl sulfate. The different isomeric forms arediagramed in FIG. 5. Thus the polymers correspond to all of the possiblecombinations of the different monomeric forms and are defined by theresidual rate of free X and Y groups.

The monomers obtained in this manner are either glucose sulfate inposition 2, 3 and/or 4 and/or glucose carboxymethyl sulfate. The sulfateis fixed either on the glucose or on the carboxylic group.

Thus, in FIG. 5, in which the bonds of the groups schematized by adotted line represent the monomers in which all of the circularcombinations can be envisaged.

1) Synthesis of carboxymethyl dextran sulfate Designated CM_(n)DS_(m)

a) Carboxymethylation

The first dextran carboxymethylation step is performed according to theprotocol described in Mauzac et al. (Mauzac et al., Anticoagulantactivities of dextran derivatives. Part I: Synthesis andcharacterization, 1984, Biomaterials 6/61-63). It comprises anetherification of the hydroxyl functions of the glucose residue of thedextran in order to obtain a carboxymethyl dextran. This reaction can bereproduced multiple times and results in products referred to as CM_(n)Din which n represents the number of carboxymethylation steps. Thevarious products referred to as CM_(n)D are characterized by anincreasing percentage of COOH. This process thus makes it possible toobtain different rates of carboxymethylation of the dextran as indicatedin the table of FIG. 6.

Thus, in a refrigerated 250-milliliter balloon flask, Dextran T40 (37.37g, 9.34 10⁻⁴ mole), from Sigma and of molecular weight 40,000 D, issolubilized (182 milliliters of distilled water) at 4° C. A sodasolution (74 g, 1.85 mole in 124 milliliters of distilled water), alsocooled to 4° C., is poured slowly into the Dextran solution whilemaintaining constant the temperature of 4° C. Monochloroacetic acid(76.2 g, 0.806 mole), reduced to a fine powder, is added slowly whilemaintaining the same reaction temperature. However, the temperature ofthe reactional medium rises at the end of the reaction from 4° C. to 21°C. within several minutes. The mixture is then brought to 50° C. in athermostated oil bath for 40 minutes. During heating, the reactionalmedium acquires a yellow coloration. After cooling, the pH isneutralized to 7.2 with glacial acetic acid (Takakura, 1990). Thecarboxymethyl dextran is collected by precipitation in cold absoluteethanol (5 to 6 times the reactional volume). It is then dried in anoven under vacuum. The polymer CM₁D is purified by ultrafiltration thenlyophilized (mass=55 g gross).

The presence of the carboxylic ions is quantified by reverse acid-basicquantification using nitric acid. The base used is 1 N soda. The resultis expressed in % of COOH groups. % COOH of CM₁D=48.98%. This percentagemeans that statistically approximately one out of every two glucoseunits was carboxymethylated.

In practice, this reaction can be performed multiple times to attain thedesired substitution rates. The same protocol was, therefore, appliedfor the synthesis of CM₂D from CM₁D and of CM₃D from CM₂D:

-   -   % COOH of CM₂D=91.8% and % COOH of CM₃D=118.3%.        b) Sulfatation

The sulfatation reaction of the residual hydroxyl functions after thecarboxymethylation steps is performed with chlorosulfonic acid. Itproduces the compounds referred to as CM_(n)DS_(m) which are presentedin FIG. 6, in which m corresponds to the chlorosulfonic acid equivalentsas defined in the example below.

Example of Sulfatation of CM₁D

Five hundred milligrams (MW=7.8 mmol/g) of carboxymethyl₁dextran (CM₁D)are dispersed in 40 milliliters of dry dichloromethane. The number ofhydroxyl residues that remain free and capable of reacting in asulfatation reaction is nOH=4·10⁻³ mole. The reaction was performed inthe presence of one chlorosulfonic acid equivalent (nClSO₃H=4·10⁻³ mole)or approximately=0.5 g or a volume of 0.3 milliliters, the density ofthe chlorosulfonic acid solution being 1.75. The 0.3 milliliters ofchlorosulfonic acid are diluted in 4 milliliters of dehydrateddichloromethane. The CM₁DS₁ obtained in this manner is recovered byfiltration of the reactional medium under vacuum on frit.

The same reaction can be performed in the presence of 0.5 or 1.5 or 2 or3 equivalents of acid chlorosulfonic acid or an excess so as to graftthe increasing quantities of O-sulfate groups. The RGTA polymers therebyobtained are referred to as CM_(n)DS_(m), with n=1, 2, etc. and m=0.5,1, 2, etc.

Using the same principle and with the goal of comparing these polymerswith other sulfated molecules, we considered as comparison products withthe CM_(n)DS_(m) either commercial dextran sulfates (Pharmacia Biotechproduct, code 17-0270-01) or dextrans sulfated from dextran T₄₀ in thepresence of m equivalents of chlorosulfonic acid, i.e., under reactionconditions comparable to those employed for producing the CM_(n)DS_(m).

The results of different quantitative determination of the X groups bytitration and the Y groups by elemental analysis of the levels of sulfuratoms make it possible to specify the corresponding values x and y foreach of the CM_(n)DS_(m) compounds synthesized. These data are presentedin FIG. 6. This figure also indicates this percentage for commercialdextran sulfate and for the dextrans sulfated under the same conditionsas the CM_(n)DS_(m).

2) Synthesis of the carboxymethyl dextran-phenyl sulfonate PolymersIndicated as CM₂DPhS and of a carboxymethyl dextran sulfate phenylsulfonate Designated CM₂DPhSS

These polymers are constituted by a sequence of motifs shown in FIG. 7and correspond to those of FIG. 4 in which Z was nothing. These polymersare constituted by a sequence of motifs of type A-X, A-Y and A-Z asshown in FIG. 6.

In this example, the group Z is phenyl sulfonate indicated as PhS. FIG.7 shows a monomer A-Z in the case in which Z received as an additionproduct a carboxyl radical grafted in position 2 of the originalglucose, which was itself sulfated in positions 3 and 4.

The polymer CM₂D (2 g; 3.90 mmol) is dissolved in 13 milliliters ofdistilled water. The pH of the solution is adjusted to 3.5 with 3 M HCl.The coupling agent EEDQ (1.93 g, 2 eq.) is dissolved in 16 millilitersof ethanol (0.12 g of EEDQ/milliliter) at 40° C. The EEDQ solution isadded progressively to the polymer and the reactional mixture isstrongly agitated for 30 minutes. The phenylsulfanilic acid salt(NH₂PhSO₃Na, 3.045 g, 2 eq.) is then added in small amounts. The pH ofthe reaction is adjusted to 9 with soda. The mixture is agitated at roomtemperature for 4 hours and then neutralized with dilute HCl. Theproduct CM₂DPhS is then ultrafiltered, evaporated under vacuum and thenlyophilized (1.66 g).

-   -   The elemental and acid-base analyses of the CM₂DPhS yielded: %        C=33.9; % H=5.28; % N=0.3; % S=0.67; % COOH=81%.    -   The elemental and acid-base titrimetric analyses of the polymer        CM₂DPhSS*₁ yielded % C=23.16; % H=3.72; % N=0.27; S=9.60; %        COOH=52%.        3) Synthesis of a carboxymethyl dextran N and O sulfate        Derivative

These polymers are constituted by a sequence of motifs of type A-X, A-Yand A-Z as shown in FIG. 6, in which one of the characteristic motifsA-Z is presented in FIG. 8. In this example, the group Z which isethylenediamine, indicated as DE, is grafted on the carboxyl radical ofthe carbon 2 of the original glucose monomer, which is itself sulfatedin positions 3 and 4.

The same protocol as employed above was applied to CM₂D with, in a firststage, the addition of an ethylenediamine group Z, indicated as DE, soas to yield the product designated CM₂DE.

-   -   The elemental and acid-base titrimetric analyses of the polymer        CM₂DE showed: % C=37.67; % H=6.25; % N=5.35; % COOH=19%.

The sulfatation protocol was performed on a fraction of this polymerusing 1 chlorosulfonic acid equivalent. The products obtained correspondto the CM₂DES₁ represented in FIG. 7. In this case, according to thegeneral formula of the RGTA, Z is diethylamine.

-   -   The elemental and acid-base titrimetric analyses of the polymer        CM₂DDES₁ showed: % C=36.83; % H=5.82; % N=4.67; % S=1.82; %        COOH=10.7%.

These syntheses make it possible to graft the N-sulfate groups inaddition to the O-sulfate groups of the CMDS. These different polymersenable evaluation of the specific roles of th N-sulfate and O-sulfategroups of the CM₂DES in relation to the phenyl sulfonate groups,indicated as PhS, of the compounds CM₂DPhS, the sulfate and sulfonatecompounds of the CM₂DPhSS or to the compounds. In this type of polymer,it would seem that the sulfate groups are essentially linked to theproperties of the RGTA.

4) Synthesis of the Polymers of carboxymethyl dextran-phenylalaninesulfate Designated CM₃DPheS and of carboxymethyl dextran-tyrosinesulfate Designated CM₃DTrS Represented in FIG. 9

These polymers are constituted of a sequence of motifs of type A-X, A-Yand A-Z as shown in FIG. 6 in which these characteristic motifs arerepresented in FIG. 9. In this example, the groups Z are respectivelyeither phenylalanine for Phe or tyrosine for Tyr. They were subjected tothe addition process on a carboxyl radical grafted on position 2 of theoriginal glucose which is itself sulfated on positions 3 and 4.

In these polymers, Z is an amino acid with either phenylalanine (Phe) ortyrosine (Tyr) and Y is an —OSO₃ ⁻ group.

These syntheses were performed so as to evaluate the importance of thearomatic structures of these two amino acids Phe and Tyr by replacingthe benzylamine indicated as B as is known in the CMBDS of the priorart, but the salting out of which in in vivo applications can bedetrimental by generating risks of tumorigenicity.

The polymer CM₃D (% COOH=136%, 0.6 g, 3.014 mmol) is dissolved in 6milliliters of distilled water. The pH of the solution is adjusted to3.5 with 3M HCl. The coupling agent EEDQ (745 milligrams, 1 eq.) isdissolved in 6.2 milliliters of ethanol at 40° C. The EEDQ solution isadded to the polymer drop by drop and the reactional mixture is agitatedvigorously for 30 minutes at room temperature. The phenylalanine methylester (1.3 g, 2 eq.) is added very slowly to the mixture and the pH isbrought from 5 to 9 with soda. The reaction is maintained at roomtemperature for 4 hours. The solution is then neutralized with diluteHCl. The polymer CM₃DPhe is then ultrafiltered, evaporated under vacuumand then lyophilized (524 milligrams).

-   -   The elemental and acid-base titrimetric analyses of the polymer        CM₃DPh showed: % C=36.9; % H=5.03; % N=0.44; % COOH=101.05%.

The same protocol was performed on the CM₃D with the tyrosine methylester CM₃DTyr.

-   -   The elemental and acid-base titrimetric analyses of the polymer        CM₃DTyr showed: % C=34.41; % H=5.12; % N=0.28; % COOH=101.76%.

The sulfatation protocol was performed on these two polymers using 2chlorosulfonic acid equivalents.

-   -   The elemental and acid-base titrimetric analyses of the polymer        CM₃DPheS*₂ showed: % C=18.08; % H=2.89; % N=0.30; % S=14.71; %        COOH=28.79.    -   The elemental and acid-base titrimetric analyses of the polymer        CM₃DTyrS₂ showed: % C=17.99; % H=3.13; % N=0.3; % S=14.35; %        COOH=19.85.        5) Synthesis of the Lipidic Polymers: Example with carboxymethyl        dextran-oleic acid sulfate (CM₁DoleicS) and carboxymethyl        dextran-palmitic acid sulfate (CM₁DpalmS) Represented in FIG. 10

The polymers are constituted by a sequence of motifs of type A-X, A-Yand A-Z as represented in FIG. 6 in which the characteristic motifs arerepresented in FIG. 10. In this example, the groups Z, which arerespectively either oleic acid indicated as oleic or palmitic acidindicated as palm, were added on the hydroxyl of the carbon 3 of acarboxymethyl glucose in position 2 sulfated in position 4.

These compounds respond to formula (I) in which Y═—OSO₃ ⁻ and Z is oleicor palmitic acid. These fatty, acids were grafted to evaluate theimportance of the hydrophobicity/hydrophilia balance of the polymers inaddition to the role itself of these fatty acids.

To the polymer CM₁D (1 g, 2.43 mmol) dissolved in DMSO (16 milliliters)was added triethylamine (0.8 milliliters) and then oleic chloride (1.6milliliters) was added drop by drop. The reactional mixture was agitatedat room temperature for 2 hours. The polymer was precipitated in 120milliliters of ethyl acetate, centrifuged and then dried under vacuum.The precipitate was dissolved in 20 milliliters of 2M sodium acetate andthe salt formed was precipitated in ethanol (160 milliliters), filtered,dissolved in 20 milliliters of distilled water then finally dialyzedagainst water. After dialysis (24 h), the solution evaporated undervacuum yielded the lipid dextran CM₁Doleic (751 milligrams).

-   -   Elemental analysis of the product CM₁Doleic: % C=34.25 and %        H=5.91.

The same protocol was performed with the palmitic chloride polymerCM₁Dpalm.

-   -   The elemental analyses of the polymer CM₁Dpalm showed: %        C=35.13, % H=5.96.

The sulfatation protocol was performed on these two polymers using 1chlorosulfonic acid equivalent.

-   -   The elemental analyses of the polymer CM₁DoleicS*₁ showed: %        C=28.28; % H=5.04; % S=5.26.    -   Elemental analyses of the product CM₁DpalmS*₁ showed: % C=29.17;        % H=4.88; % S=5.14.

The different examples presented which involve grafting a group Z yieldthe compounds, the definitions of several of which are presented inTable II below.

TABLE II Reference Groups X Y Z RGTA 1110 CM₂DPhSS₁ 52.1 43.8 8.9 RGTA1111 CM₂DES₁ 10.7 42.4 21.2 RGTA 1112 CM₂DPheS₂ 28.9 56.2 17.9 RGTA 1113CM₃DTyrS₂ 19.8 65.9 28.9 RGTA 1114 CM₁DpalmS₁ 39.8 47.4 3.8 RGTA 1115CM₁DolcicS₁ 36.0 43.9 2.2

Table II above indicates the percentages of substitution of the polymerscontaining a group Z.

6) Purification Steps for the Different Dextran Derivatives of the AboveExamples

a) Dialysis to Equilibrium

After each synthesis step the polymers are collected in solid form(precipitation or filtration followed by lyophilization). The polymersare then resolubilized in the minimum volume of distilled water and thenintroduced into dialysis tubing (Spectrapor) with a cut-off threshold of6000 to 8000 g/mole⁻¹. The dialysis is performed against twice-distilledwater (MilliQ) in a ratio of 1 volume of product per 50 volumes of waterfor 4 to 5 days with two changes of water per day.

b) Chromatography

The preceding step is associated with HPLC chromatography on molecularsieve (Column TSK) in order to establish the molar masses of thepurified polymers.

c) Tangential Ultrafiltration

After the dialysis, the content of the tubing is ultrafiltered in anultrafiltration cell (Pellikon, Millipore) on a cellulose membrane witha cutoff threshold of 10,000 g/mole⁻¹. The quality of the purificationwas monitored with a conductimetry cell. When the conductivity of thewater eliminated at the outlet of the cell had returned to theconductivity of pure distilled water (2 μS), the purification wasstopped and the solution was concentrated prior to lyophilization.

7) Determination of the Percentages of Substitution

For the two groups of examples presented above, the percentages ofsubstitution of the groups X, Y and possibly Z were determined in thefollowing manner.

a) Poly β-malic acid Polymers (Examples 1)

For these polymers, the percentages of substitution are defined a prioriin relation to the proportion of the different monoesters subjected topolymerization.

a) Dextran Polymers (Examples 2)

Two cases must be envisaged for these polymers obtained from dextrandepending on the definition of the group Z.

The first case corresponds to the case of the CM_(n)DS_(m) in whichZ=nothing; the second case depends on the chemical nature of Z.

On each glucose residue, three hydroxyl functions are capable ofreacting. A relative molar mass of 54 g/mole⁻¹ is attributed to eachhydroxyl function, i.e., one third of the molecular mass of 162 g/mole⁻¹of a constitutive residue of dextran, It is assumed that each hydroxylhas the same reactivity and that the substitutions first affect eachglucose unit once prior to a possible second substitution on the sameresidue.

A dextran T 40 of 40,000 g/mole⁻¹ thus contains 247 glucose residues ofmolar mass 162 g/mole⁻¹.

The substitution rates attained during the carboxymethylations aredetermined by acid-base determination with an automatic titrimeter(Tacussel). This determination finds a value x₁ corresponding to thenumber of moles of acid fixed per gram of polymer.

Thus, when a hydroxyl is substituted, there appears on the glucose amotif: —OCH₂COONa. Each of these substituted subunits has a relativemolecular mass of 240 g/mole⁻¹.

Multiple motifs appear after sulfatation.

The rates of free carboxylic groups determined by acid-basedetermination gives a value X₂ which is always lower than the initialvalue X. The difference X₁−X₂ corresponds to the motifs —OCH₂COO—SO₃Na.Each of these substituted subunits has a molecular mass of 320 g/mole⁻¹.

NMR analysis revealed that the S corresponds to a sulfatation of thefree hydroxyls of the glucose residues in addition to the precedingreaction. In this case, a motif —OSO₃Na appears. Each of these sulfatedglucose subunits has a relative molecular mass of 200 g/mole⁻¹. Themicroanalyses provide the rates of S as a percentage of the mass of thepolymer.

It is, therefore, possible at the end of synthesis to obtain thepercentages of free carboxyl radicals X₁ and X₂ determined respectivelybefore and after the sulfatation step, taking into account that thepolymer contains:

-   -   a unsubstituted glucose residues of mass 162 g.    -   X₂ free carboxylic residues of mass 240 g.    -   X₁−X₂ sulfated carboxylic residues of mass 320 g.    -   Y sulfated glucose residues of mass 200 g.

Based on these data, it is possible to establish the percentage ofsubstitution of the groups X and Y.

Thus, the percentage of sulfur provided by the microanalysis results (S%) make it possible to determine the number of atoms of sulfur (Σ_(S))grafted on the polymer. This number of atoms is Σ_(S)=(S %×MM)/32×100,in which 32 is the atomic mass of S and MM is the molar mass of thesynthesized polymer.

It is possible to obtain from this the percentage Y of radicals SO₃ ⁻ asequal to: (100×S %×MM)/247×3200.

In the second case in which the grafted group Z is for example tyrosine,the same reasoning is applicable with the value of nitrogen given by theelemental analysis results.

B) Properties of the Polymers of the Invention

The properties that are common to the RGTA and the HBGFPP

-   -   1) They are devoid of significant anticoagulant activity, i.e.,        they present an activity lower than 50 IU/milligram compared to        that of heparin whose activity is on the order of 150 to 170        IU/milligram.    -   2) They stabilize and potentiate the growth factors that present        an affinity for heparin, particularly as examples FGF1 and/or        FGF2 and/or TGFβ.    -   3) They protect these factors against proteolytic agents such as        trypsin.    -   4) They inhibit the protease activities implicated in the        inflammatory process such as for example leukocyte elastase or        plasmin.    -   5) They exert a cicatrizing effect in at least one of the models        presented in the cited patents, i.e., the muscles, the nerves or        the gastrointestinal tract.        The novel properties of the RGTA    -   6) They protect and potentiate the enzymatic activities        implicated in combating oxidative stress such as for example        superoxide dismutase or SOD. Due to this property, they act as        antioxidant agents and can be used alone or associated with SOD        in the therapeutic and/or cosmetic indications of SOD or as an        antioxidant protective agent especially in the protection of        foods and nutrients.    -   7) They inhibit the activity of enzymes such as calpaine.    -   8) They inhibit the activity of enzymes such as heparitinase or        heparinase.    -   9) They increase the survival of cells subjected to ionizing        radiation and regulate the secretion on both the quantitative        and qualitative levels of the constituents of their matrix as        well as the collagens for example.    -   10) They act as antifibrotic agents by modulating the growth of        the mesenchymal cells such as the smooth muscle cells, the        fibroblasts or the hepatic cells and the quality of the type of        collagen that they secrete.    -   11) They present a slow degradability, a criterion which enables        their differentiation from the heparan sulfates which are        products that are naturally degraded by heparinase or        heparitinase.    -   12) They neither contain nor liberate after degradation products        that are known to be toxic, such as for example can occur with        the CMDBS with the grafted groups Z=benzylamine, which therefore        rules out the CMDBS.    -   13) They present in vivo capacities of protection and tissular        regeneration in the following different models:        -   a) cutaneous wound lesion,        -   b) regeneration of osseous tissues such as the long bones,            with the example of the femur,        -   c) protection against the loss of osseous tissue and            regulation of its reorganization such as in the case of            osteoporosis or periodontal disease. They are, therefore,            regulator agents of tissular homeostasis and of tissular            masses such as osseous or muscular mass.        -   d) hepatic regeneration or protection against the            degeneration of the central and peripheral nervous systems,        -   e) protection against the deleterious effects of ischemia            regardless of its localization.

EXAMPLE 3 Measurement of the Anticoagulant Activities of the Polymers ofExamples 1 and 2

The coagulation tests were performed using the Activated Cephalin Timetechnique or A.C.T. (Biggs, 1972, In: Human Blood Coagulation, OxfordBlackwell Scientific Publications). One hundred microliters of a polymersolution at different concentrations in Owen Koller buffer are incubatedfor 5 minutes at 37° C. with 100 microliters of plasma poor in plateletsand 100 microliters of a solution of rabbit brain cephalin. 100microliters of 0.25 M calcium chloride are added and the time untilappearance of the coagulum is referenced by chronometry.

As shown in FIG. 11, the polymers of Examples 1 and 2 do not presentanticoagulant activities greater than 50 IU/milligram, especially withrespect to heparin which was used as a positive control. It should benoted that all of the values for the anticoagulant activities of theproducts presented here as examples are lower than 10 IU/milligram ofproduct.

EXAMPLE 4 Stabilization and Potentiation of the Polymers of Examples 1and 2 on Growth Factors Presenting an Affinity for Heparin andParticularly as Examples FGF1 and/or FGF2 and/or TGFβ

This example considers the effect of the polymers on the stabilizationof FGF1 and the effect on the potentiation of FGF1 and FGF2. Theseeffects are evaluated on the growth of 3T3 BALB/c or CCL39 cells. Theconditions employed for these experiments are those described in theprior art in the patents relative to the HBGFPPs which are incorporatedin the present invention by reference.

1) Effects of the Polymers of the Invention on the Stabilization of FGF1or FGF2

FIG. 12 shows the effects of the RGTA against thermal degradation at 20°C. and at 37° C. in relation to the incubation time of FGF2 conservedalone or in the presence of the tested products. The ED₅₀ represents theconcentration in micrograms/milliliter of FGF1, here 6nano-grams/milliliter, that must be inoculated in a culture offibroblastic cells, the cells CCL 39, in order to obtain 50% of themaximum rate of incorporation of tritiated thymidine.

The results obtained show that all of the polymers tested exert at 20°C. as well as at 37° C. protective effects comparable to those ofheparin, and with greater efficacy in some cases.

2) Effects of the Polymers of the Invention on the Potentiation of FGF1or FGF2

The protocol is the same as described previously with a variablequantity of FGF possibly associated with a constant quantity of polymer.The concentration of polymer employed corresponds to that whichpotentiates to the maximum the mitogenic effect of the FGF. These testsare performed on 3T3 cells for FGF1 and FGF2. The controls are the sameas those previously cited with the exception of a systematicdetermination for each test of the ED₅₀ value.

The molecules of these two families of tested polymers potentiate theactions of FGF1 and FGF2 because ED₅₀ values are obtained for FGF valuesthat are lower or comparable to that obtained in the presence of heparin(FIG. 13).

Example 5 Protection of the Factors Against Proteolytic Agents such asTrypsin

Trypsin is a proteolytic enzyme with a broad spectrum of action which isused in in vitro tests and which is one of the primordial functionalenzymes in the digestive process.

The test for protection against trypsin was therefore performed. Onenanogram/final milliliter of iodinated FGF2 or 5 micrograms/finalmilliliter of trypsin are incubated in a first step for 15 minutes at37° C. with different concentrations of polymers (0.5 to 500micrograms/milliliter) in a 100 mM Tris HCl buffer, 0.18 M NaCl, Brij0.03% pH 7.6. Five micrograms/final milliliter of trypsin or 1nanogram/final milliliter of iodinated FGF2 are then added respectively.The total reactional volume is 30 μl. The enzymatic reaction is stoppedafter 2 h of incubation at 37° C. by addition of Laemmli buffer andheating for 5 minutes at 90° C. (Laemmli U.K. Cleavage of structuralproteins during assembly of the head of the bacteriophage T4, Nature,1970, 227: 680-685).

Each sample is placed on a 15% polyacrylamide gel. Migration isperformed for 1 h at 200 V in ice. The gels are dried for 2 h at 80° C.under vacuum and exposed at −80° C. in the presence of anautoradiographic film. The intensity of the bands is measured by imageanalysis and the percentage of FGF2 protected in relation to percentageof FGF2 degraded is calculated.

Protection of FGF1 and TGFβ

FIG. 14 shows the protective effects expressed as % of protection of thepoly-β-malic acid polymers on FGF1, FGF2 and TGFβ in relation to anattack by trypsin. FIG. 15 presents the protective effects expressed in% of protection of the polymers derived from dextran on FGF2 and TGFβ inrelation to an attack by trypsin.

Most of these polymers exert a protective effect which is comparable tothat of heparin which was used as positive reference.

EXAMPLE 6 Inhibition of the Protease Activities Implicated in theInflammatory Process such as for Example Leukocyte Elastase or Plasmin

Leukocyte elastase and plasmin are key proteases in the installation andunfolding of the inflammatory tests. These tests are intended toestablish whether the polymers protect the growth factors fromdegradations by human leukocyte elastase.

The tests for protection against leukocyte elastase are thereforeperformed. The protocol employed is the same as that used with trypsinwith 30 nanograms/final milliliter of elastase and 0.5 to 500micrograms/milliliter of polymers. The buffer is 100 mM Tris HCl, 0.18 MNaCl, 0.03% Brij pH 8. The intensity of the bands is evaluated by imageanalysis and the percentage of nondegraded FGF2 is calculated. Thepositive control for these tests is heparin. Dextran T40 and dextransulfate were used as internal references.

FIG. 16 presents the inhibitory effects of the different polymersexpressed by their IC₅₀ values.

EXAMPLE 7 Example of the Effects of the RGTA on the Regeneration,Protection and Functional Restoration of the Tissues: Case of theRegeneration of Skeletal Muscle

The model employed for evaluating most effectively the cicatrizingproperties of the RGTA was that of the crushed muscle as defined andpresented in French Patent No. 2 718 026.

After crushing the EDL (Extensor Digitorum Longus) of the rear paw ofthe rat and injection of the crushed muscle into a solution ofphysiological serum containing or not containing the test substances,the muscles treated in this manner are recovered 8 days after theoperation. Analysis of the weights as well as a histological study makepossible quantification of the effects of the polymers on muscularregeneration. The results are expressed in % in relation to thecharacteristics of a muscle that only received an injection of thephysiological serum without polymer under the same experimentalconditions. FIG. 17 presents the results obtained which demonstrate thatthe new polymers derived from the dextran skeleton as well as thecopolymers of β-malic acid exert the claimed effects.

It is important to note that these effects on muscular regeneration areobtained not only by in situ injection of the polymers but also byintravenous, intra-arterial or intramuscular injection as long as thedoses injected are selected in relation to the routes of administration.

EXAMPLE 8 Effects of the RGTA on the Regeneration of Flat Bone

The model employed for evaluating the cicatrizing properties of the RGTAis the method already known in the prior art and described by BlanquaertF, et al., Bone, 1995; 17(6): 499-506.

This model comprises performing a circular trepanation of 5 millimetersin diameter in the calvaria of an adult rat. The defect is filled with acollagen buffer that has been cut to the same dimensions and impregnatedwith or not impregnated with a solution containing the RGTA. In theexample presented here, the polymers studied are the type CMS polymers(RGTA 1005 and 1012) and the β-malic acid copolymers (RGTA 2011). TableIII below presents the percentages of osseous filling established byimage analysis of the radiographs taken 35 days after treatment.

TABLE III Type of treatment % of osseous filling control 18 ± 4.8 RGTA2011 56 ± 7.0 RGTA 1005 54 ± 6.9 RGTA 1012 72 ± 8.9

Thus, both types of polymers stimulate osseous regeneration since underthe cicatrization conditions the sagittal suture forms ad integrum.

EXAMPLE 9 Protection and Potentiation of the Enzymatic ActivitiesImplicated in Combating Oxidative Stress such as for Example SuperoxideDismutase or SOD

The production of O₂ ⁻ ions and that of hydrogen peroxide (H₂O₂)represent radicals that exert especially destructive cytotoxic effects.SOD or superoxide dismutase are enzymes engaged in the detoxification ofthese radicals. They are agents that preserve the organism fromoxidative stress.

The RGTA present various types of activity in relation to SOD:

-   -   They exert a potentiating effect on the catalytic activity of        SOD at neutral pH and a protective and potentiating effect at        acid and basic pH values.    -   They present the property of protecting SOD in relation to        enzymatic degradations such as for example trypsin and also in        relation to heat treatment.    -   On models of activated monocyte cultures, they stimulate the        catalytic activity of the endogenous SOD and enable diminishment        of the production of the superoxide ions.

In all cases, the quantitative determination of SOD activity isperformed using Pick's technique (Freund M and Pick E, The mechanism ofaction of lymphokines. IX. The enzymatic basis of hydrogen production bylymphokine-activated macrophages. J Immunol 1986, 15; 137(4):1312-1318). This technique is based on the determination of the O₂ ⁻ions by reduction of cytochrome c. MnSOD (Sigma ref. S 8151) at 30U/milliliter is dissolved in the presence of different RGTA at theconcentration of 10 micrograms/milliliter. These mixtures are subjectedto different treatments.

The SOD activity is evaluated in the presence of differentconcentrations of polymers under normal reactional conditions.

The mixtures of SOD and polymers are either subjected at roomtemperature to the action of trypsin (same conditions as for theprotection tests of Example 5) or they are subjected to thermaltreatment at 60° C. for 30 minutes. The residual catalytic activity ofthe SOD of these mixtures is then evaluated by conventional enzymatictechniques or on a cellular system.

The samples treated in this manner are incubated in a suspension ofmonocytes (2.5·10⁶ cells/milliliter) stimulated by 200 nM of PMA. Thiscondition induces the production of superoxide anion by these monocytesactivated into macrophages. The stimulation by PMA induces an increasein the production of superoxide ions normally produced at a basal levelin the absence of activation. The addition of active MnSOD used aspositive control diminishes the quantity of superoxide ions produced.

Under these conditions, the lower the production of superoxide ions, thehigher will be the residual catalytic activity of the SOD contained inthe mixtures. Thus, these tests make it possible to evaluate theprotective and potentiator effects of the polymers on the activity ofexogenous as well as endogenous SOD.

FIG. 18 illustrates the protective and potentiator effects of the RGTAon the catalytic activity of SOD in vitro at different pH values. FIG.19 illustrates the protective effects of the RGTA on SOD subjected to anattack by trypsin or after a thermal shock. FIG. 20 illustrates thepotentiator effects of the SOD activity on the production of superoxideions by the activated macrophages.

Conclusion: Thus, the polymers exert potentiator and protective effectsin both acellular and cellular systems. Thus, the addition of differentpolymers modulates the catalytic activity of SOD added endogenously aswell as SOD produced endogenously by the cells.

EXAMPLE 10 Inhibition of the Activity of Enzymes such as Calpaine by theRGTA

1) Introduction

Calpaine 1 (Sigma P 4533) is used in this example. Thus, 0.78 enzymeunit (57.2 nM) are incubated in 1 milliliter of 50 mM Tris-HCl buffer,pH 7.4, 0.5% Brij, 2 mM CaCl₂, 2 mMDT and 0.15M NaCl. The RGTA solutionsto be tested are added over 5 minutes at 27° C. The substrate (250micromolar of N-succinyl-leu-tyr-amido-7-methyl coumarin, from Sigma (S1153) is then added to a quartz tank to which the mixture describedabove is then added. Measurements are then performed every 3 to 5minutes by excitation at 380 nm and detection in fluorescence at 460 nmof the transformation of the substrate into 7-amino-4-methyl coumarin(according to the protocol described by Sasaki, Kikuchi, Yumoto N,Yoshimura and Murachi T, 1984, in J. Biol. Chem. 259 (20), p12489-12494).

The results obtained are summarized in FIG. 21.

2) Conclusion

We can predict from the results obtained in the inhibition of theactivity of calpaine by the RGTA that the RGTA have a protectiveactivity against the lesions induced by cerebral ischemia as could beanticipated from the publications of Markgraf C G et al. (Stroke, 1998,29, 152-158) or Saido et al. (Neuroscience Letters, 1997, 16; 227:75-78) which describe the effect of calpaine inhibitors such as MDL28170or the protein calpastatine in the treatment of postischemic lesions ofthe cortex or hippocampus. Administration of RGTA via the local orintravenous route has the effect of inhibiting the calpaines andpromoting the repair of the nervous tissue which has been injuredespecially by lack of oxygen supply as is the case in ischemia.

EXAMPLE 11 Inhibition of the Activity of Enzymes such as Heparitinase bythe RGTA

a) Inhibitory Activity of Heparinase and Heparitinase

Measurement of the inhibition by the RGTA of heparinase or heparitinaseactivities is performed using as substrate heparan sulfates radio-taggedwith sulfur 35 and present in an extracellular matrix synthesized byendothelial cells cultured in the presence of Na2 35SO4 for 7 days. Theprotocol used is the one described by Ishai-Michaeli Ret al. (Importanceof size and sulfation of heparin in release of basic fibroblast growthfactor from the vascular endothelium and extracellular matrix.Biochemistry 1992; 31(7): 2080-2088). The extracellular matrix obtainedafter elimination of the endothelial cells is then incubated for 24 h at37° C. in the presence or absence of heparinase and 0.5 microgram/ml ofRGTA. The incubation medium is collected and deposited on a Sepharose6.B column in accordance with the protocol described in Ishai-Michaeli(Biochemistry 1992; 313(7): 2080-2088) to measure the degradation ofradio-labeled heparan sulfates. FIG. 22 illustrates the resultsobtained. The heparan sulfates of high molecular weight corresponding tothe material not degraded by the enzyme heparinase were eluted first(fractions 3 to 20). The heparinase treatment caused the disappearanceof this peak and the appearance of a peak corresponding to low molecularweight fractions of degraded heparan sulfates (fractions 20 to 40). Inthe example presented, the effect of RGTA 1005 at 50 micrograms/mlinduces 50% inhibition and 100 micrograms/ml induces 100% inhibition ofthe heparinase activity.

EXAMPLE 12 Effects of the RGTA on the Protection of Human IntestinalSmooth Muscle Cells Subjected to Ionizing Radiation; Effects on theirSurvival and on their Antifibrotic Effects Evaluated by Means of theQuantity and Quality of the Secreted Collagens

The formation of a fibrous or fibrotic tissue is an essentialphysiological step associated with the processes of tissue repair andrestructuring. The fibrotic tissue is a filling tissue, normallytransitory, intended to conserve the structural and functional integrityof the tissues and organs. It is characterized by its richness incollagens of the extracellular matrix.

When this condition persists or develops, it corresponds to a pathologythat illustrates a disturbance of the structural and functionalhomeostasis of the tissues and is manifested by an abnormally highaccumulation of extracellular matrix which generates a fibrosis. Afibrosis, regardless of its origin, is characterized by:

-   -   the presence of a permanent inflammatory infiltrate, the        existence of a disequilibrium in the balance between a        proliferative and a quiescent state of the conjunctival or        mesenchymal cells such as fibroblasts or smooth muscle cells,        the progressive destruction of the invaded tissue which is        renewed only slightly or in a defective manner, the existence of        a disequilibrium of the balance between the synthesis and the        degradation of the extracellular matrix.

A fibrosis can be induced either subsequent to a trauma of variousorigins (infectious, mechanical, toxic, etc.) or subsequent to ionizingradiation (notably by γ rays). In this case, we are dealing withradioinduced fibroses as is frequently the case in patients undergoingradiotherapy.

The collagens are the major components of the extracellular matrix ofnormal tissues as well as of fibrotic tissues. The collagens areessentially synthesized by the mesenchymal cells such as the fibroblastsand the smooth muscle cells. In a fibrous tissue, the total collagen isquantitatively increased due to reasons affecting on both thequantitative and qualitative levels the synthesis and/or degradation ofthe collagens, i.e., the dynamic of the restructuring. Fibroses arecharacterized by an increase in type III collagen, with this takingplace preferentially in the case of radioinduced fibroses. This increasein type III collagen is associated with an increase but to a lesserdegree in the ratio between type III collagen and type I collagen.Another collagen, type V collagen, is associated with the quality of theorganization of the collagen fibers in the matrix, i.e., thefibrillogenesis. In fibrotic tissues, the decline in the levels of typeV collagen is one of the origins of the loss of structure of thecollagen fibers of the extracellular matrix.

The cellular model employed in this example is that of HISM cells, HumanIntestinal Smooth Muscle cells (American Type Culture Collection,Rockville, Md. ATCC CRL 192), stemming from the muscularis propria ofhuman jejunum (Graham M., Diegelmann R., Elson C., Bitar K. and EhrlichH., Proc. Soc. Exp. Biol. Med. 176 (1984) 503). This line of humanintestinal smooth muscle cells was used to evaluate the effects ofradiation on cellular survival and the induction of fibrotic phenomenaanalyzed by means of the quantity and quality of the types of collagensecreted by these normal cells or in an inflammatory situation or in therepair process by a fibrosis.

The cells are cultured in DMEM medium containing 1 g/l of glucose, 1% ofL-glutamine, 1% penicillin-streptomycin and 10% of fetal calf serum andconserved in an incubator at 37° C. under an atmosphere saturated at 5%CO₂ and 95% relative humidity, in 75-cm² plates. The HISM cells areseeded on plates with 24 flat-bottom wells at the rate of 20,000 cellsper well. The volume of each well is brought up to 2 milliliters ofmedium. Various growth kinetics are implemented in the presence or lackof presence of RGTA at different concentrations ranging from 0.4 to 400micrograms/milliliter.

Irradiation is performed from a ⁶⁰cobalt irradiation source in anincubator in which the culture plates are arranged. Two plates aresubjected simultaneously to this irradiation the source of which isvertical and the doses are evaluated in relation to the surfaceirradiated. The irradiator flow rate is 1 Gy/min. The doses absorbed are10 Gy for an irradiation time per plate of 10 minutes.

Different protocols are used to evaluate the role of the RGTA dependingon whether they are added to the culture medium before, during or afterthe irradiation, i.e., at the preventive or curative level, or both atthe same time. Table IV below provides more specific information on thedifferent protocols.

TABLE IV Sequence RGTA Irradiation RGTA Control — — — Irradiation — 0 —Curative — 0 0 Preventive 0 0 — Preventive and curative 0 0 0

The preventive effect is evaluated by addition of RGTA (+) at the doseof 400 micrograms/ml in the culture medium 48 hours prior to theirradiation. The curative effect is evaluated by addition of RGTA 2hours after irradiation at the same doses as for the preventive effect.For the cumulative preventive and curative effects, the cells arecontinuously cultured in the presence of the same doses of RGTA.

72 hours after the irradiation, the cells are incubated in a medium freeof serum in the presence of tritiated proline (10 microCi/ml) andascorbic acid (50 micrograms/ml) for 24 hours. The supernatant is thencollected and the cellular layer is recovered using a robber-policemanin a final volume of 18 ml. The culture media and the recovered cellularlayers are extensively dialyzed (cutoff threshold of 6 to 8 kDa) againstflowing water (24 hours at 4° C.) to eliminate the small molecules fromthe macromolecules. After dialysis, aliquot fractions are collected andhydrolyzed (6M HCl, 105° C., 24 h) for determination of theradioactivity of the hydroxy(³H)proline, specific marker of thecollagens. At this stage, an aliquot fraction is used for quantificationby counting of the radioactivity of the total synthesis of thecollagens.

The remaining volume is dialyzed again in the presence of pepsin andcollagen I against 0.5 M acetic acid for 24 h at 4° C. The pepsin willdigest the noncollagenic contaminants which will be eliminated in thedialysate in the form of peptides. Type I collagen is added to augmentthe proportion of collagen which is low in each sample and to trendtowards an enzyme/substrate ratio of 1:5. The reaction conditions are asfollows: 1 ml of pepsin solution (0.5 M)+1 ml of solution of collagen 1(0.5 M)+0.514 ml of acetic acid to have a final concentration of aceticacid of 0.5M in an 18-ml sample. Each dialysate obtained in this manneris lyophilized in the cold state (−50° C.) and conserved at −20° C.until use.

The different collagen a chains are separated by polyacrylamide gelelectrophoresis in the presence of SDS (Sodium Dodecyl Sulfate) inaccordance with Leammli's method, after reduction withβ-mercaptoethanol. The radioactivity incorporated in each collagen isdetermined by hydrolysis of the α chains obtained by cutting off the gelbands corresponding to each collagen chain of a specific type. Thesebands are then dissolved in oxygenated water at 60° C. and theradioactivity contained in the different dissolved bands is counted withthe liquid scintillation β counter or by direct autoradiography on thegels.

The electrophoretic separation is performed with 5 microliters ofcollagen V and 50 microliters of each reduced sample.

These gels allow various types of processing:

-   -   Determination of the different types of collagen by analysis of        the incorporated radioactivity.    -   Quantification of these different types of collagen by        densitometric analysis after autoradiography.

The aliquot fractions collected after the dialysis against flowing waterare hydrolyzed (6M HCl, 105° C., 24 h) in sealed ampoules. The aceticacid is then evaporated, and then the content of the ampoule isresuspended in 1 ml of distilled water. The proline and hydroxyprolineare separated by the method of Rojkind and Gonzales (Rojkind M andGonzalez E, An improved method for determining specific radioactivitiesof proline-14C and hydroxyproline-14C in collagen and noncollagenousproteins. Analytical Biochemistry 1974; 57(1): 1-7). The principle isbased on oxidizing the proline and hydroxyproline with chloramine T. Theproline is transformed into pyrroline carboxylate which is soluble intoluene whereas the hydroxyproline is transformed into water-solublecarboxylate. After treatment, the proline and hydroxyproline fractionsare recovered for each sample and quantified by measurement of theradioactivity.

Under normal conditions, the HISM cells are characterized by thequantity of collagen synthesized (FIG. 23) and especially on thequalitative level by the phenotype of the collagens secreted (FIG. 24).

Under normal conditions, the collagen that comprises the majority of thecollagen is type I collagen. Type III and type V collagens are presentin smaller proportions. The quantity of type V collagen is associatedwith the quality of the organization of the fibrillogenesis.

Type III collagen is a “warning” collagen, theoretically synthesized ina transitory manner in the case of reaction to a stress but in apermanent manner in the case of a fibrosis. The proportional quantity oftype III collagen in relation to type I collagen increases in anoteworthy manner in situations involving response to a tissular lesion,stress or aggression such as for example ionizing radiation. Type IIIcollagen becomes preponderant in the matrices of tissues presenting anacute as well as a chronic fibrotic reaction. This collagen can beconsidered to be a signaling component of the fibrotic reaction.

When the HISM cells are cultured under control conditions, i.e., withoutRGTA, they synthesize these three types of collagen (FIG. 24).Irradiation modifies the quantity (FIG. 23) and the quality (FIG. 24) ofthe collagens produced. The overall synthesis of collagen increases byclose to 50% (FIG. 23). Secretion of this type I collagen and especiallyof type III collagen increases considerably (respectively by 50% andclose to 500%). In contrast, synthesis of type V collagen diminishes bymore than 50%.

The presence of different RGTA (RGTA 1005 and RGTA 1025) restores almostexactly the control behavior of the cells despite their exposure to theirradiation. FIG. 23 shows that the overall synthesis of collagenreturns to normal values especially for the conditions of preventive orcumulative (preventive+curative) treatments. FIG. 24 confirms thisreturn to a reference homeostasis especially in the case of cumulatedtreatments. The curative as well as the preventive treatments exert thesame types of effects, especially with regard to the values of theratios of secretion of type I and type III collagens which return tovalues comparable to those of the control cells. These RGTA restore thefunctioning of the HISM cells in relation to the collagens.

The RGTA also act on the survival of the cells (FIG. 25). In fact,irradiation induces a high cellular mortality rate of more than 50% ofthe irradiated population. In the presence of the polymers, a clearprotective effect is recorded because the mortality rate is reduced toapproximately 25%, with the most pronounced effect being obtained forthe cumulated preventive+curative treatments.

EXAMPLE 13 Action as Antifibrotic Agents in Modulating the Growth ofMesenchymal Cells such as Smooth Muscle Cells, Fibroblasts or HepaticCells and the Quality of the Type of Collagen that they Secrete

As discussed in example 12 above, fibrosis manifests an alteration ingrowth of the mesenchymal cells associated with a modification of thequantity and quality of the collagens synthesized.

This example uses another cellular model which comprises smooth musclecells from the pig aorta. These cells are obtained by means of the aortaexplant method. The aorta is composed of three cellular layers: theadventitia (external layer) which contains the fibroblastic cells, themedia (central layer) which contains the smooth muscle cells (SMC) andthe intima (internal layer) which contains the endothelial cells.

The media is removed and torn up into tiny pieces. The explants arecultured on a 25-cm² plate containing DMEM 1 g/l of glucose with phenolred, with the addition of 20% of fetal calf serum (FCS), 1% ofL-glutamine and 1% of penicillin-streptomycin. The cells become detachedfrom these explants and colonize the medium (stage P0). Two weeks afterthe beginning of the culture phase, the cells are congealed at acellular density of 2 million cells in 10% DMSO and DMEM 20% FCS.

The growth kinetics are determined by evaluating the number of cells perwell by means of an automatic particle counter (Coulter Counter ZM,Coultronics) which was previously calibrated by an evaluation of thecell diameter by means of a Malassez cell. Each counting was performedon an average of 4 wells from which the cells were detached by means of500 microliters/well of a trypsin-EDTA (10 mM) solution.

The smooth muscle cells were seeded under the same experimentalconditions as the HISM cells. The growth kinetics were determined in thepresence of or lack of presence of polymers of the series RGTA 1000 to1025 for the polymers in which Z is nothing, and of the series RGTA 1110to 1115 for the polymers in which Z exists, and of heparin as control,at different concentrations ranging from 0.4 to 400 mg/ml.

The percentage of inhibition of the proliferation is calculatedaccording to the following formula:I %=100×(1−net growth with polymers/net control growth)in which net growth represents the difference between the quantity ofcells counted when they merge with the number of cells seeded at thebeginning of culturing.

The control corresponds to the smooth muscle tissue cultures in theabsence of polymers. Heparin and the RGTA represent the differenteffectors tested which are capable of modifying the biological activityof the cell.

Under the same conditions as employed for the HISM cells, the synthesisand typing of the collagen by the smooth muscle cells was determined.

FIG. 26 shows the results obtained. The RGTA exert an inhibitory effecton the proliferation of the smooth muscle cells which is comparable tothat of heparin which was used as control reference molecule. Thiseffect can be seen from the values in the two first columns. However,and in contrast to heparin, these polymers reestablish the collagensecretion phenol-type. In summary, the overall synthesis rate ofcollagens is significantly diminished in the presence of RGTA 1005, 1012and 1013. At the qualitative level, these same polymers, again incontrast to heparin, diminish the rate of synthesis of type I and typeIII collagens while increasing the secretion of type V collagen.

These effects confirm the antifibrotic properties of the polymers of theinvention.

EXAMPLE 14 Effects of the RGTA on Regeneration of the Skin in the Rat

This example illustrates the effect of the RGTA on deep cutaneouscicatrization after suture in the rat.

Male Hairless rats weighing 250 grams were anesthetized by an IMinjection of ketamine and Largactil. Two 3-cm excisions were madelaterally in relation to the dorsal axis of symmetry of the animals oneach side of the spinal column (FIG. 27). In relation to the controlanimals which were not treated with RGTA 1012, the treated animalsreceived via the intramuscular route an injection of a solution of RGTA1012 at 1 milligram per kilogram 30 minutes prior to the excision and100 microliters of a solution at 100 micrograms per milliliter astopical application on the wound just prior to suturing. The suture wasperformed in a single plane with an intradermal continuous suture withProlene 2-0. The treated rats and the control rats weremacrophotographed on days 7, 21 and 60 after the operation. At each ofthese time points, three rats from each experimental series wereeuthanized so as to be able to perform a histological study of thecutaneous cicatricial samples.

FIG. 27 shows the effects of the RGTA on the cutaneous cicatrization:

-   -   FIG. 27-A: Deep cutaneous incision performed down to the        subjacent muscular floor, 3 cm long, on both sides of the spinal        column.    -   FIG. 27-B: Dorsal view of the animal after suturing of the wound        edges by continuous suture.    -   FIG. 27-C: Appearance of the cicatrices ten days after the        treatment. C1 and C2 correspond to a control animal treated with        physiological serum without RGTA 1012. In C1 the threads have        not been removed in contrast to photograph C2. The cicatrix is        visible and still presents scabs of coagulated blood especially        where traces can be seen of the needles used for the suture. C3        and C4 correspond to an animal treated with physiological serum        containing RGTA 1012. At the same time, the cicatrices are no        longer visible. The suture threads are visible in figure C3        whereas they were removed in figure C4. In this photograph, only        a fine border marks the original incision.    -   FIG. 27-D: Histological analysis. D1 and D2 present the        histological sections of the skin that was incised 60 days        earlier. D1 corresponds to a control animal that was treated        with physiological serum without RGTA 1012; figure D2        corresponds to an animal treated with physiological serum        containing RGTA 1012. In D1, the dermis subjacent to a still        imperfectly mature epithelium has not returned to a structure        identical to that of normal skin. The restructuring is very        partial. The opposite is true of D2 in which the skin has        returned to a structure and an organization identical to that of        skin that was never injured. The only trace of a cicatrix is        visible in the depth of the dermis where an incompletely        restructured zone can be detected. In this later case, there is        complete absence of external cicatrix.

The photographs of FIG. 27 show at the end of 7 days the disappearanceof the superficial cicatrix whether the threads had (C4) or had not (C3)been removed whereas in the control animals a cicatrix can be seen underboth of these conditions (C1 and C2).

A histological analysis performed at 60 days revealed that the skins ofthe untreated animals (D1) presented a dermis that was absolutely notmature whereas the animals treated with the RGTA revealed only a slightdermal trace at the deep level. The histology of the skin of the treatedanimals (D2) has an appearance comparable to the skin of a controlanimal not subjected to any cicatricial processes whatsoever. In thismodel, the RGTA not only accelerate the cicatrization rate of thecutaneous floor but also and especially enable a restoration of thecicatricial tissue which results in a regenerated tissue in which notrace of fibrosis can be detected. This example shows that the RGTA areespecially powerful regulators of tissular homeostasis.

EXAMPLE 15 Protective Effects Against Ischemia Manifested by the RGTA

This example demonstrates, using the polymer RGTA 1005, the protectiveeffects of the RGTA against tissular damage which enabled conservationof 80% of the mass of an organ compared to the untreated organs (FIG.28). These protective effects of the RGTA against the deleteriouseffects induced by the stress caused by a lack of oxygen supply stemmingfrom an ischemia of the muscles are presented in the experimentationdescribed below.

The model employed is inspired by the model described by Hansen-Smith,F. M., Carlson, B. M. & Irwin, K. L. (1980, Revascularization of thefreely grafted extensor digitorum longus muscle in the rat. Am. J. Anat.158, 65-82). The experimental procedure consists of sectioning theneurovascular trunk of the EDL muscles (Extensor Digitorum Longus) onthe two rear paws of adult Wistar rats (350 g) at the level of its entryin the muscle and of completing the ischemia by a ligature of the twotendons. An injection of 100 microliters of an RGTA 1005 solution at 50micrograms per milliliter in physiological serum was then made directlyinto an EDL muscle. The same volume of physiological serum without RGTAwas injected into the other contralateral muscle.

Seven days after the injection, the muscles were removed and examinedwith a microscope after histological preparation. In each group oftreated or untreated muscles, groups of parameters such as the meandiameter of the muscle, the thickness of the epimysium, of theperipheral zone and the mean diameter of the ischemic zone were measuredusing a 10× objective and a micrometric scale. The number of layers ofmuscle fibers that survived the ischemia in the peripheral zone wascounted in thirty different fields selected at random and observed witha 20× objective.

FIG. 28 shows the protective effects of the RGTA (RGTA 1005) againsttissue injury in a muscle ischemia model in the rat. FIG. 28-A and FIG.28-B show, respectively, histological muscle sections from a control rat(28-A) and from a rat treated (28-B) with a solution of RGTA 1005. Inthe control, the ischemia caused the degeneration of the muscle fiberswith the exception of a corona of peripheral fibers in contact with theepimysium. Administration of RGTA 1005 limits to a considerable degreethe degeneration of the deep muscle fibers just as it diminishes in avery significant manner the inflammatory reaction and the degradationthat this reaction induces. Thus, RGTA 10015 protects the cells from thedeleterious effects induced by ischemia.

It can be seen in FIG. 28 that after one week, the mean diameter isunchanged after treatment with RGTA (5.2±0.3 mm) compared to thediameter (5.1±0.2 mm) of an uninjured control muscle. The inflammatoryreaction in the epimysium is diminished in the muscles treated with theRGTA in a very significant manner since the thicknesses of the epimysiaare 10±5 micrometers with treatment by the RGTA compared to 85=10micrometers without treatment with the RGTA (p<0.01). The centralischemic zone of the EDL muscles not treated with RGTA presents a meandiameter of 4.4000±100 micrometers in which the muscle fibers havecompletely disappeared. This zone is surrounded by a peripheral zone of270±50 micrometers containing an average of 3.2±0.5 layers of musclefibers. The treatment with the RGTA is characterized by a clear decreasein the size of the central degenerated zone whose mean diameter is300±200 micrometers (p<0.05) and an augmentation of the peripheral zonethe thickness of which is 700±40 micrometers (p<0.05) and which contains8.3±1.8 layers (p<0.01) of fiber.

EXAMPLE 16 Effects of the RGTA on the Regeneration of Long Bone

This example illustrates the reconstruction of an osseous defect createdin the diaphysial shaft of a rat femur, restored to the original stateafter 8 weeks and better at 12 weeks, with reconstitution of a medullarycavity identical to the original one and mature cortices as in theoriginal unfractured bone (FIG. 29).

Male Wistar rats (Ico: WI (IOPS AF/Han), Iffa Credo) weighing from 275to 325 grams were used. The study was performed in accordance with theEEC recommendations on animal experimentation (decree 87-848—Apr. 19,1987). The animals were anesthetized by injection of sodiumpentobarbital. The femur was approached laterally. The muscle andperiosteal tissues were separated from the diaphysial shaft. Ahigh-density polyethylene plate was fixed to the surface of the femurwith Kirschner pins. A segmentary defect of 5 millimeters wasimplemented in the middle of the femoral diaphysis. An implant wasinserted in the place of the osseous defect prior to suturing thetissues. This implant corresponds to a demineralized allogenic osseousmatrix prepared from femoral diaphyses from other rats according to theprocedure described by F. Blanquaert et al. (1995, Bone, 17: 499-506),which were impregnated or not impregnated with RGTA 1012 by incubationin a saline solution comprising 100 micrograms per milliliter of thisproduct. The animals were then maintained in cages without ambulatoryrestraints. The femurs of the animals were radiographed every two weeksfor 12 weeks before being euthanized. The femurs were then collected andsubjected to the treatments required for histological study. Theradiographs were studied especially in their densitometric aspects byimage analysis.

FIG. 29 shows the effects of the RGTA on the regeneration of the longbones and shows especially the histological and radiographic studies offemurs from rats which were either treated or not treated by RGTA 1015.

FIG. 29-A shows the model defect created in the femoral diaphysis.

FIGS. 29-B, 29-D, 29-F and 29-H represent radiographs of femurs fromdifferent experimental groups. FIGS. 29-C, 29-E and 29-G representhistological sections of operative pieces corresponding to the specimenspresented in 29-D, 29-F and 29-H, respectively.

FIGS. 29-C and 29-D show the femoral defect which did not receive anyparticular treatment. During the 12-week period there was no cicatricialphenomenon and the osseous shaft was not reconstituted.

FIGS. 29-E and 29-F show that the osseous defect was filled by thedemineralized osseous matrix. In this case, filling took place but noreorganization can be seen. The structure of the filling does notpresent the organization of a traditional long bone.

In FIGS. 29-G and 29-H, it can be seen that the osseous defect wasfilled by the demineralized osseous matrix impregnated in a solution ofRGTA 1015. In this case, the structure of the filling tissuescorresponds to that of normal bone. The compact cortical bone iscomparable to the uninjured zone in which the mark of the fixation screwcan be seen. This part delimits a cavity filled with bone marrow incontinuity with the original medullary cavity.

FIGS. 29-I1, 29-I2, 29-J1 and 29-J2 show the effect of RGTA 1015 on thereformation rate of the long bone. Radiographs I1 and I2 were taken at 8weeks; the radiographs J1 and J2 were taken at 12 weeks. I1 and J1correspond to the treatment presented in 29-E and 29-F in which theosseous defect was filled solely by the demineralized osseous matrixwithout RGTA. I2 and J2 correspond to the treatment presented in 29-Gand 29-H in which the osseous defect was filled by the demineralizedosseous matrix impregnated in a solution of RGTA 1015. These radiographsshow an acceleration of the cicatrization and especially of thematuration of the reformed bones and in terms of a pronouncedcorticalization (I2 and J2) which can not be detected in I1 and J1. RGTA1015 acts as a regeneration agent which enables acceleratedreconstitution of the osseous structure of long bone with a structureidentical to that of the original bone.

Thus, in a surprising manner, RGTA 1012 impregnated in the demineralizedosseous matrix, compared to a matrix impregnated in physiological serumwithout the polymer, induces an extremely significant acceleration inthe restructuring and maturation processes of the bone. This effect ismanifested in the appearance of new cortices after only 8 weeks whereasthis phenomenon did not take place under the control condition. After 12weeks, six of the seven animals treated by the association with RGTA1012 presented in the radiological study the evidence of a completeunion of the defect with the reformation of thick and delimited corticeswhereas without CM₁DS₂ only the union is observable with radiologicalimages of immature bone without corticalization. A quantitative imageanalysis study of the radiographs confirmed these observations. Thisstudy also showed that the profile of the bones treated by RGTA 1012 iscomparable to that of normal bone, with the only difference being thatthe osseous material has a relatively low density at the experimentaltime point of 12 weeks. Projection of the density of the bone that isnewly formed in the original defect shows that the treatment by thepolymer RGTA 1012 induces a tissular restructuring via the corticalreformation and a medullary cavity.

The histological studies correlate with and confirm the resultsestablished on the basis of image analysis of the radiological data.These histological studies demonstrate the presence of new corticesconstituted of compact bone with still several regions of marrow and thepresence of a medullary cavity in continuity with the originaldiaphysial shaft, filled with new bone marrow. The femurs treatedwithout RGTA 1012 do not show any figure of maturation. Thereconstituted bone does not present any specific organization. It isconstituted by a heterogeneous mixture of compact bone and medullarytissue without specific territorial delimitation.

These results illustrate the osteoinductive effects of the polymers ofthe invention on their capacity to induce not only the repair of thelong bones but also and especially their potential to regulate thehomeostasis of the regenerated tissues at the level of their mass aswell as their reorganization.

The properties of the RGTA as regulatory agent of the homeostasis of theosseous tissues, i.e., of the tissular mass, its functionality and itsrestructuring are confirmed by Example 17 below.

EXAMPLE 17 Effects of the RGTA on the Restructuring and the Protectionof the Osseous Mass in a Model of Acute Periodontal Disease in theHamster

The model used in this example pertains to the osseous restructuring ofthe mandible of the hamster.

Periodontal disease is induced in golden Syrian hamsters (Dépré breedingcenter, references HSM 41/50) after two weeks of a hyperglucidic diet.The feed administered was composed of sucrose (56%), powdered skimmedmilk (28%), whole wheat flour (6%), brewer's yeast (4%), powderedalfalfa (3%), liver powder (1%) and sodium chloride (2%).

The animals were distributed into experimental groups. Fourteen animalsconstituted the control groups which received a normal diet comprised ofdry feed. Twenty-four animals constituted the experimental groupssubjected to the hyperglucidic diet. They developed chronic periodontaldisease which was established at the end of two months. After this timepoint, the experimental groups received each week for 3 weeks anintramuscular injection of RGTA 1005 at different doses comprisedbetween 0.1 and 15 milligrams per kilogram in a volume of 0.5millimeters of buffered physiological serum. The control groups receivedan injection of physiological serum without RGTA 1005 (SHAM). Theanimals' weights were measured each week. One month after each type oftreatment, the animals were sacrificed, the mandibles were collected andprepared for histological study. The inclusions of the operative pieceswere implemented in stabilized methyl methacrylate.

In this model of periodontal disease, only 200 micrometers in heightcould be processed for each hemimandible. The system is standardized toalways study the same sequence of sections at a depth that is definedand referenced by the osseous tissues between the roots of the two firstmolars of the lingual side.

The periodontal disease at the level of these molars is manifested bythe appearance of a periodontal pouch which delimits a volume filledwith bacterial plaque. This disease leads to a notable destruction ofthe periodontal bone which is characterized by a notable osteoclasticresorption and a reduction in the osseous surfaces in apposition, i.e.,to an osteosynthesis phase.

The osseous resorption is quantified by measuring the zones of contactbetween the bone and the osteoclasts (Oc). These are giant cells thatare stained blue by toluidine blue. The apposition in turn ischaracterized by a band of osteoid tissue, identified by an attenuatedblue coloration covered by osteoblasts. In contrast to the osteoclasts,the osteoblasts are cells of small size, of cubic form and mononuclear.The quantifications of these phenomena are performed with an imagingsystem that uses an image-processing program.

FIG. 30 presents the quantifications obtained under the variousexperimental conditions. In the control animals who did not develop thedisease, the resorption activity is practically nonexistent whereas theapposition activity is noteworthy. In the untreated animals (SHAM), thedisease was strongly developed and characterized by an intenseresorption and a low degree of apposition. In the animals treated by theCM₁DS₂, particularly at the dose of 1.5 milligrams per kilogram, theresorption rate is greatly diminished and is associated with a verylarge apposition rate which reaches close to 80% of the rate observed inthe control animals.

These effects were obtained by intramuscular injection. They show thatthe RGTA regulate the osseous tissue mass by re-equilibrating thebalance of the restructuring between resorption and apposition.

EXAMPLE 18 Pharmacokinetic Data Demonstrating the Properties of the RGTAfor Vectorizing Molecules Towards Injured Tissues

This example illustrates the capacity presented by the polymers of theinvention for fixing themselves in a specific manner and concentratingat the level of the sites of tissular injury.

In the model of the regeneration of crushed muscle described in Example7, a lesion is implemented on the EDL of the left paw of the animals.Each animal receives an intravenous injection of 2·10⁶ cpm of RGTA 1012radiolabeled with tritium by the company SibTech, Inc. (NY, USA). Thespecific activity of this tracer is 20 millicurie per milligram.

At different postoperative times, the injured EDL muscles of the leftpaws and the uninjured muscles of the right paws were collected andfrozen in liquid nitrogen. The implementation of frozen histologicalsections enabled, by means of a beta imager (Societe Biospace),measurement of the quantity of radiolabeled product fixed at the levelof the tissue sections studies. The results presented in Table V belowshow that the injured muscles concentrated after 24 hours a quantity ofradioactive product approximately 5 times greater than the uninjuredmuscles the labeling level of which did not differ from the device'sbackground noise.

TABLE V cpm of RGTA 1012 fixed at the level of the tissues Postuperativctime 24 hours 48 hours 96 hours Injured muscle tissue 11,800 ± 890   10,150 ± 10120 9000 ± 790 Contralateral control 2060 ± 530 1980 ± 3901870 ± 640 muscle tissue Background noise 1800 ± 160 1780 ± 210 1950 ±180 of registration

These results demonstrate the autotargeting capacities of the polymersof the invention which concentrate themselves specifically at the levelof the tissues presenting a disorder or a lesion. Thus, a particularlyinteresting property of these polymers resides in their capacity tovectorize a medical or diagnostic principle.

EXAMPLE 19 Effects of RGTA in Periodontics and on the Osseous Mass

In the field of periodontics, a macroscopic study of the loss ofalveolar bone was performed on the periodontitis of the hamster.

After a 2-month period for induction of the disease, the animals (n=20)were treated via the IM route for an additional 2 months without actingon the initial cause of the disease, i.e., the bacterial component.Other animals were left untreated (n=20). These two groups were comparedwith healthy hamsters (no periodontitis) (n=12).

At the end of the experimental period, all fleshy tissues were removedfrom the superior maxillaries so as to enable determination of the boneloss. The zone of the first molar was photographed in a standardizedmanner. A reference line was traced on each photograph whichcorresponded to the enamel-cement junction. Then a second line wastraced which ran along the contours of the osseous ridge. These twolines were reunited in front of and behind the first molar. This surfacewas measured.

It should be noted that in the controls, there is a zone of denudationof the root which corresponds to:

-   -   a zone of physiological fibrous insertion which anchors the gum        on the root,    -   a loss of bone height which is produced in relation to the aging        of the animals.

In our animals this denudation represented 0.96 mm².

In the diseased animals, the bone loss was 1.34 mm², which includes theinitial physiological zone (which was destroyed over the course of theperiodontitis) and a part of the loss due to aging. Nevertheless, wecould conclude that the disease induced a bone loss on the order of 0.38mm² (difference: p<0.0001).

In the treated animals, there is always an incompressible zone (the sameis true of the controls). The denudation of the root represents 1.02 mm²in these animals. Thus, there is a net deficit in relation to thecontrols of 0.06 mm² (difference not significant) and an improvement by0.32 mm² in relation to the untreated animals (p=0.0005).

The invention claimed is:
 1. A process for treating and/or preventinglesions and diseases induced by an oxidative stress and oxidizing agentscomprising administering, to a patient in need thereof, atherapeutically effective amount of a pharmaceutical composition whichcomprises at least one biocompatible polymer of the following generalformula (I): A_(a)X_(x)Y_(y) wherein: A represents a monomer, Xrepresents a carboxyl group bonded to monomer A and is contained withina group according to the following formula: —R—COO—R′, in which R is abond or an aliphatic hydrocarbon chain, optionally branched and/orunsaturated, and which can contain one or more aromatic rings except forbenzylamine and benzylamine sulfonate, and R′ represents a hydrogen atomor a cation, Y represents a sulfate or sulfonate group bonded to monomerA and is contained within a group according to one of the followingformulas: —R—O—SO₃—R′, —R—N—SO₃-R′, —R—SO₃—R′, in which R is a bond oran aliphatic hydrocarbon chain, optionally branched and/or unsaturated,and which can contain one or more aromatic rings except for benzylamineand benzylamine sulfonate, and R′ represents a hydrogen atom or acation, a represents the number of monomers A such that the mass of theat least one biocompatible polymer of formula (I) is greater thanapproximately 5,000 da, x represents the substitution rate of themonomers A by the groups X, which is between approximately 20 and 150%,and y represents the substitution rate of the monomers A by the groupsY, which is between approximately 30 and 150%, and a pharmaceuticallyacceptable vehicle.
 2. A process according to claim 1, wherein R of thegroups X and Y is selected from the group consisting of alkyl, allyl,aryl, linear and branched groups.
 3. A process according to claim 1,wherein the monomers A, which can be identical or different, areselected from the group consisting of sugars, esters, alcohols, aminoacids and nucleotides.
 4. A process according to claim 1, wherein atleast one monomer A is a sugar.
 5. A process according to claim 1,wherein the at least one biocompatible polymer comprises at least onefunctional chemical group Z, which is different from X and Y, and whichconfers supplementary biological or physicochemical properties, whereinthe at least one functional chemical group Z is conjugated to A and/or Xand/or Y.
 6. A process according to claim 5, wherein the at least onefunctional chemical group Z comprises a plurality of functional chemicalgroups Z, and wherein the rate of substitution of monomers A by theplurality of functional chemical groups Z is between approximately 0 and50%.
 7. A process according to claim 5, wherein the at least onefunctional chemical group Z comprises a plurality of functional chemicalgroups Z, and wherein at least one of the plurality of functionalchemical groups Z is a substance which confers on the at least onebiocompatible polymer additional solubility or lipophilic properties. 8.A process according to claim 7, wherein the plurality of functionalchemical groups Z, which can be identical or different, are selectedfrom the group consisting of amino acids, fatty acids, fatty alcohols,ceramides, and nucleotide addressing sequences.
 9. A process accordingto claim 5, wherein the at least one functional chemical group Zcomprises a plurality of functional chemical groups Z, and wherein theplurality of functional chemical groups Z, which can be identical ordifferent, are therapeutic or diagnostic agents.
 10. A process accordingto claim 9, wherein the at least one functional chemical group Z isselected from the group consisting of an anti-inflammatory,antimicrobial, antibiotic, enzyme and a growth factor.
 11. A processaccording to claim 5, wherein the at least one functional chemical groupZ is covalently bonded directly to the monomers A or covalently bondedto the X and/or Y groups.
 12. A process according to claim 5, whereinthe at least one functional chemical group Z is conjugated to the atleast one biocompatible polymer of formula (I) by bonds other thancovalent bonds.
 13. A process according to claim 12, wherein the groupsat least one functional chemical group Z is conjugated to the at leastone biocompatible polymer of formula (I) by ionic or hydrophilicinteraction.
 14. A process according to claim 1, wherein the at leastone biocompatible polymer comprises at least one functional chemicalgroup Z, which is different from X and Y, and which conferssupplementary biological or physicochemical properties, wherein thegroups X, Y and Z are bonded directly to the monomer A, or bonded toeach other with only one of them being bonded to the monomer A.